Antibiotic formulations, unit doses, kits, and methods

ABSTRACT

An aqueous or powder composition includes anti-gram-negative antibiotic or salt thereof being present at an amount ranging from about 100 mg/ml to about 200 mg/ml. Another aqueous or powder composition includes anti-gram-positive antibiotic or salt thereof being present at a concentration ranging from about 0.6 to about 0.9 of the water solubility limit, at 25° C. and 1.0 atmosphere, of the anti-gram-positive antibiotic or salt thereof. Other embodiments include unit doses, kits, and methods.

BACKGROUND

This application is a continuation of U.S. application Ser. No.11/529,128, filed Sep. 28, 2006, which claims priority to U.S.Provisional Application No. 60/722,564, filed Sep. 29, 2005, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to anti-infective, such as antibioticformulations, unit doses, kits, and methods, and in particular toaminoglycoside formulations, unit doses, kits, and methods

2. Background of the Invention

The need for effective therapeutic treatment of patients has resulted inthe development of a variety of pharmaceutical formulation deliverytechniques. One traditional technique involves the oral delivery of apharmaceutical formulation in the form of a pill, capsule, elixir, orthe like. However, oral delivery can in some cases be undesirable. Forexample, many pharmaceutical formulations may be degraded in thedigestive tract before the body can effectively absorb them. Inhalabledrug delivery, where a patient orally or nasally inhales an aerosolizedpharmaceutical formulation to deliver the formulation to the patient'srespiratory tract, may also be effective and/or desirable. In oneinhalation technique, an aerosolized pharmaceutical formulation provideslocal therapeutic treatment and/or prophylaxis to a portion of therespiratory tract, such as the lungs, to treat respiratory diseases suchas asthma and emphysema and/or to treat local lung infections, such asfungal infections and cystic fibrosis. In another inhalation technique,a pharmaceutical formulation is delivered deep within a patient's lungswhere it may be absorbed into the bloodstream for systemic delivery ofthe formulation throughout the body. Many types of aerosolizationdevices exist including devices comprising a pharmaceutical formulationstored in or with a propellant, devices that aerosolize a powder,devices which use a compressed gas or other mechanism to aerosolize aliquid pharmaceutical formulation, and similar devices.

One known aerosolization device is commonly referred to as a nebulizer.A nebulizer imparts energy into a liquid pharmaceutical formulation toaerosolize the liquid, and to allow delivery to the pulmonary system,e.g. the lungs, of a patient. A nebulizer comprises a liquid deliverysystem, such as a container having a reservoir that contains a liquidpharmaceutical formulation. The liquid pharmaceutical formulationgenerally comprises an active agent that is either in solution orsuspended within a liquid medium. In one type of nebulizer, generallyreferred to as a jet nebulizer, compressed gas is forced through anorifice in the container. The compressed gas forces liquid to bewithdrawn through a nozzle, and the withdrawn liquid mixes with theflowing gas to form aerosol droplets. A cloud of droplets is thenadministered to the patient's respiratory tract. In another type ofnebulizer, generally referred to as a vibrating mesh nebulizer, energy,such as mechanical energy, vibrates a mesh. This vibration of the meshaerosolizes the liquid pharmaceutical formulation to create an aerosolcloud that is administered to the patient's lungs. In still another typeof nebulizer, ultrasonic waves are generated to directly vibrate andaerosolize the pharmaceutical formulation.

Nebulizers are often used to deliver (1) an aerosolized pharmaceuticalformulation to a hospitalized or non-ambulatory patient; (2) large dosesof aerosolized active agent; and/or (3) an aerosolized pharmaceuticalformulation to a child or other patient unable to receive a dry powderor propellant based pharmaceutical formulation.

Nebulizers are useful for delivering an aerosolized pharmaceuticalformulation to the respiratory tract of a patient who is breathing underthe assistance of a ventilator. But there are problems associated withthe introduction of aerosolized pharmaceutical formulation intoventilator circuits. For example, by introducing the aerosolizedpharmaceutical formulation into the inspiratory line of the ventilator,significant residence volume exists between the point of introductionand the patient's lungs. Accordingly, large amounts of aerosolizedpharmaceutical formulation are needed and much of the formulation islost to the exhalation line. This problem is exacerbated when thenebulizer is used in conjunction with ventilators having continual biasflows. In addition, the large residence volume in the ventilator linemay dilute the aerosolized pharmaceutical formulation to an extent wherethe amount delivered to the patient is difficult to reproduceconsistently.

U.S. Published Application Nos. 2004/0011358, 2004/0035490, and2004/0035413, which are incorporated herein by reference in theirentireties, disclose methods, devices, and formulations for targetedendobronchial therapy. Aerosolized antibiotics are delivered into aventilator circuit. The aerosol generator, e.g., nebulizer, may beplaced in the lower part of a Y-piece, for example, distal to the Y, tobe proximal to a patient airway and/or endotracheal tube.

U.S. Pat. Nos. 5,508,269 and 6,890,907, which are incorporated herein byreference in their entireties, disclose aminoglycoside solutions fornebulization. The '269 patent discloses that if the solution approachesthe solubility of tobramycin, 160 mg/ml, precipitation on storage isexpected. The '269 patent also discloses that a higher concentration oftobramycin than is clinically needed is economically disadvantageous.Further the '269 patent discloses that a more concentrated solution willincrease the osmolarity of the solution, thus decreasing the output ofthe formulation with both jet and ultrasonic nebulizers. The '269 patentdiscloses that the alternative of a more concentrated solution in asmaller total volume is also disadvantageous. The '269 patent furtherdiscloses that most nebulizers have a dead space volume of 1 ml, i.e.,that of the last 1 ml of solution is wasted because the nebulizer is notperforming. Therefore, while for example, a 2 ml solution would have 50%wastage, the 5 ml solution (the capacity of the nebulizer) has only 20%wastage. Additionally, the '269 patent discloses that since there is nosufficient aerosolization of the drug into the small particles, the drugin large particles or as a solution is deposited in the upper airwaysand induces cough and may also cause bronchospasm. According to the '269patent, large aerosol particles also limit the drug delivery

There remains, however, a need for improved antibiotic formulations,such as antibiotic formulations for nebulization. There also remains aneed for improved unit doses and kits of antibiotic formulations.Accordingly, there also remains a need for improved methods of makingand/or using such antibiotic formulations.

SUMMARY OF THE INVENTION

Accordingly, one or more embodiments of the present invention satisfiesone or more of these needs. Thus the present invention providesantibiotic formulations, such as antibiotic formulations fornebulization. The present invention also provides unit doses and kits ofantibiotic formulations. The present invention further provides methodsof making and/or using such antibiotic formulations. Other features andadvantages of the present invention will be set forth in the descriptionof invention that follows, and will be apparent, in part, from thedescription or may be learned by practice of the invention. Theinvention will be realized and attained by the devices and methodsparticularly pointed out in the written description and claims hereof.

In one aspect, one or more embodiments are directed to an aqueouscomposition, comprising an antibiotic or salt thereof being present at atherapeutic-effective (including prophylatic-effective) amount. In oneor more embodiments, the therapeutic-effective amount is based uponaerosolized administration to the pulmonary system.

In one aspect, one or more embodiments are directed to an aqueouscomposition, comprising anti-gram-negative antibiotic or salt thereofbeing present at an amount ranging from about 90 mg/ml to about 300mg/ml.

In another aspect, an aqueous composition comprises anti-gram-negativeantibiotic or salt thereof, and optionally, a bronchodilator.

In still another aspect, an aqueous composition comprisesanti-gram-positive antibiotic or salt thereof being present at aconcentration ranging from about 0.6 to about 0.9 of the watersolubility limit, at 25° C. and 1.0 atmosphere, of theanti-gram-positive antibiotic or salt thereof.

In yet another aspect, a unit dose comprises a container and an aqueouscomposition, comprising anti-gram-negative antibiotic or salt thereofbeing present at a concentration ranging from about 100 mg/ml to about200 mg/ml.

In still another aspect, a kit comprises a first container containing afirst aqueous solution comprising an anti gram-negative antibiotic orsalt thereof; and a second container containing a second aqueoussolution comprising an anti gram-negative antibiotic or salt thereof. Aconcentration, or an amount, or both of the first aqueous solution isdifferent from a concentration, or an amount, or both, of the secondaqueous solution.

In yet another aspect, a kit comprises a first container containing afirst aqueous solution comprising anti gram-negative antibiotic or saltthereof, and a second container containing a second aqueous solutioncomprising anti gram-positive antibiotic or salt thereof.

In another aspect, a unit dose comprises a container and a powdercomprising an antibiotic or salt thereof, wherein the powder is presentin an amount ranging from about 550 mg to about 900 mg.

In still another aspect, a unit dose comprises a container; and a powdercomprising an antibiotic or salt thereof, wherein the powder is presentin an amount ranging from about 150 mg to about 450 mg.

In yet another aspect, a kit comprises a first container containing afirst composition comprising anti-gram-positive or an anti gram-negativeantibiotic or salt thereof and a second container containing a secondcomposition comprising water. The first composition and/or the secondcomposition comprises osmolality adjuster.

In another aspect, a kit comprises a first container containing a powdercomprising anti-gram-positive antibiotic or salt thereof and a secondcontainer containing a powder comprising anti gram-positive antibioticor salt thereof. A concentration, or an amount, or both, of the antigram-positive antibiotic or salt thereof in the first container isdifferent from a concentration, or an amount, or both, of the antigram-positive antibiotic or salt thereof in the second container.

In a further aspect, a kit comprises a first container containing asolution comprising anti gram-negative antibiotic or salt thereof and asecond container containing a powder comprising anti gram-positiveantibiotic or salt thereof.

In still another aspect, a method of administering an antibioticformulation to a patient in need thereof comprises aerosolizing anantibiotic formulation to administer the antibiotic formulation to thelungs of the patient. The antibiotic formulation has a concentration ofantibiotic or salt thereof ranging from about 90 mg/ml to about 300mg/ml.

In another aspect, a method of administering an antibiotic formulationto a patient in need thereof comprises inserting a tube into a tracheaof a patient. The method also comprises aerosolizing an antibioticformulation to administer the antibiotic formulation to the lungs of thepatient. The antibiotic formulation consists essentially ofanti-gram-negative antibiotic or salt thereof and water.

In yet another aspect, a method of administering an antibioticformulation to a patient in need thereof comprises aerosolizing anantibiotic formulation to administer the antibiotic formulation to thelungs of the patient. The antibiotic formulation comprises an antibioticor salt thereof at a concentration ranging from about 0.7 to about 0.9of the water solubility limit, at 25° C. and 1.0 atmosphere, of theantibiotic or salt thereof.

In a further aspect, a method of administering an antibiotic formulationto a patient in need thereof comprises dissolving an antibiotic or saltthereof in a solvent to form an antibiotic formulation, wherein theantibiotic or salt thereof is present at a concentration ranging fromabout 0.6 to about 0.9 of the water solubility limit, at 25° C. and 1.0atmosphere, of the antibiotic or salt thereof. The method also includesaerosolizing the antibiotic formulation to administer the antibioticformulation to the lungs of the patient.

In yet another aspect, a method of administering an antibioticformulation to a patient in need thereof comprises dissolving anantibiotic or salt thereof in a solvent to form an antibioticformulation. The method also includes aerosolizing the antibioticformulation to administer the antibiotic formulation to the lungs of thepatient, wherein the aerosolizing is conducted within about 16 hours ofthe dissolving.

In another aspect, a method involves forming a powder comprising anantibiotic or salt thereof. The method includes dissolving an antibioticor salt thereof in a solvent to form a solution having a concentrationranging from about 60 mg/ml to about 120 mg/ml. The method also includeslyophilizing the solution to form the powder.

In another aspect, a method involves forming a powder comprising anantibiotic or salt thereof. The method comprises dissolving anantibiotic or salt thereof in a solvent to form a solution having avolume ranging from about 4.5 ml to about 5.5 ml. The method alsoincludes lyophilizing the solution to form the dry powder.

In another aspect, any method which comprises forming a powder may alsoinclude a method of reconstituting the powder to form a liquid.Similarly any method which comprises forming a liquid comprising anantibiotic (such as a solution) may also include a method of removingthe liquid to yield a powder.

In another aspect, any two or more of any of the foregoing features,aspects versions or embodiments are combined.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described in the description ofinvention that follows, in reference to the noted plurality ofnon-limiting drawings, wherein:

FIG. 1A illustrates components of a pulmonary drug delivery systemaccording to embodiments of the present invention.

FIG. 1B shows an embodiment of a device that can be used in a pulmonarydrug delivery system according to embodiments of the invention.

FIG. 2A shows an exemplary off-ventilator configuration of a pulmonarydrug delivery system according to embodiments of the invention.

FIG. 2B is a schematic view of a pharmaceutical delivery device of oneor more embodiments of the present invention, useful for delivery ofaerosolized medicaments.

FIG. 3 shows total drug recovered (nebulizer+filters) for gentamicin asa function of fill mass and solution strength.

FIGS. 4 a-b show emitted dose of gentamicin as a function of solutionstrength and fill volume, after nebulization (FIG. 4 a) for 15 minutes,and (FIG. 4) 30 minutes.

FIG. 5 shows gentamicin residual dose retained in a nebulizer as afunction of fill volume and solution strength.

FIG. 6 shows distribution of nebulized vancomycin (60 mg/ml solution innormal saline) as a function of fill volume.

FIG. 7 shows emitted dose as a function of solution strength and fillvolume, for the case of vancomycin solution in 0.45% saline.

FIG. 8 shows emitted dose as a function of solution strength and fillvolume, for the case of vancomycin solution in water for injection(WFI).

FIG. 9 shows volume median diameter for nebulized gentamicin as afunction of solution strength and fill volume.

FIG. 10 shows cumulative particle size distributions for gentamicin atdifferent solution strengths and nebulizer fill volumes.

FIG. 11 shows volume median diameter for nebulized vancomycin (solutionin WFI) as a function of solution strength and fill volume.

FIG. 12 shows cumulative particle size distributions for nebulizedvancomycin (solution in WFI) at different solution strengths andnebulizer fill volumes

FIG. 13 shows volume median diameter for nebulized vancomycin (60 mg/mlsolution in normal saline) as a function of nebulizer fill volume.

FIG. 14 shows volume median diameter for nebulized vancomycin (solutionin 0.45% saline) as a function of solution strength and fill volume.

FIG. 15 shows volume median diameter for antibiotic drug and placebosolutions.

FIG. 16 is a graph showing amikacin stability over time (as % relatedsubstance) for a formulation according to one or more embodiments of thepresent invention, wherein the formulation was stored at three differentstorage conditions.

DESCRIPTION OF THE INVENTION

Unless otherwise stated, a reference to a compound or component includesthe compound or component by itself, as well as in combination withother compounds or components, such as mixtures of compounds.

As used herein, the singular forms “a,” “an,” and “the” include theplural reference unless the context clearly dictates otherwise.

Reference herein to “one embodiment”, “one version” or “one aspect”shall include one or more such embodiments, versions or aspects, unlessotherwise clear from the context.

“Mass median diameter” or “MMD” is a measure of mean particle size,since the powders of the invention are generally polydisperse (i.e.,consist of a range of particle sizes). MMD values as reported herein aredetermined by centrifugal sedimentation, although any number of commonlyemployed techniques can be used for measuring mean particle size.

“Mass median aerodynamic diameter” or “MMAD” is a measure of theaerodynamic size of a dispersed particle. The aerodynamic diameter isused to describe an aerosolized powder in terms of its settlingbehavior, and is the diameter of a unit density sphere having the samesettling velocity, generally in air, as the particle. The aerodynamicdiameter encompasses particle shape, density and physical size of aparticle. As used herein, MMAD refers to the midpoint or median of theaerodynamic particle size distribution of an aerosolized powderdetermined by cascade impaction.

Anti-gram negative, and gram-negative antibiotic are usedinterchangeably to refer to antibiotic active agents (and formulationscomprising such active agents) which have effectiveness against gramnegative bacteria. Similarly, anti-gram positive, and gram-positiveantibiotic are used interchangeably to refer to antibiotic active agents(and formulations comprising such active agents) which haveeffectiveness against gram positive bacteria.

“Antibiotic” moreover includes anti-infectives, such as antivirals andantifungals, as well as antibiotics, unless the context indicatesotherwise.

“Pharmaceutic formulation” and “composition” may be sometimes usedinterchangeably to refer to a formulation comprising an antibiotic.

As an overview, in one or more embodiments, an aqueous compositioncomprises anti-gram-negative and/or anti-gram positive antibiotic orsalt thereof being present at an amount ranging from about 100 mg/ml toabout 200 mg/ml.

In one or more embodiments, an aqueous composition comprises anantibiotic or salt thereof, and bronchodilator.

In one or more embodiments, an aqueous composition comprises anantibiotic or salt thereof being present at a concentration ranging fromabout 0.6 to about 0.9 of the water solubility limit, at 25° C. and 1.0atmosphere, of the antibiotic or salt thereof.

In one or more embodiments, a unit dose comprises a container and anaqueous composition, comprising an anti-gram-negative antibiotic or saltthereof at a concentration ranging from about 100 mg/ml to about 200mg/ml.

In one or more embodiments, a kit comprises a first container containinga first aqueous solution comprising anti-gram-negative antibiotic orsalt thereof; and a second container containing a second aqueoussolution comprising anti-gram-negative antibiotic or salt thereof. Aconcentration, or an amount, or both, of the first aqueous solution isdifferent from a concentration, or an amount, or both, of the secondaqueous solution.

In one or more embodiments, a kit comprises a first container containinga first aqueous solution comprising anti-gram-negative antibiotic orsalt thereof, and a second container containing a second aqueoussolution comprising anti-gram-positive antibiotic or salt thereof.

In one or more embodiments, a unit dose comprises a container and apowder comprising an antibiotic or salt thereof, wherein the powder ispresent in an amount ranging from about 550 mg to about 900 mg.

In one or more embodiments, a unit dose comprises a container; and apowder comprising an antibiotic or salt thereof, wherein the powder ispresent in an amount ranging from about 150 mg to about 450 mg.

In one or more embodiments, a kit comprises a first container containinga first composition comprising an anti-gram-positive or an antigram-negative antibiotic or salt thereof and a second containercontaining a second composition comprising water. The first compositionand/or the second composition comprises an osmolality adjuster.

In one or more embodiments, a kit comprises a first container containinga powder comprising an anti-gram-positive antibiotic or salt thereof anda second container containing a powder comprising an anti-gram-positiveantibiotic or salt thereof. A concentration, or an amount, or both, ofthe anti-gram-positive antibiotic or salt thereof in the first containeris different from a concentration, or an amount, or both, of theanti-gram-positive antibiotic or salt thereof in the second container.

In one or more embodiments, a kit comprises a first container containinga solution comprising an anti-gram-negative antibiotic or salt thereofand a second container containing a powder comprising anti-gram-positiveantibiotic or salt thereof.

In one or more embodiments, a method of administering an antibioticformulation to a patient in need thereof comprises aerosolizing anantibiotic formulation to administer the antibiotic formulation to thepulmonary system of the patient. The antibiotic formulation has aconcentration of anti-gram-negative antibiotic or salt thereof rangingfrom about 100 mg/ml to about 200 mg/ml.

In one or more embodiments, a method of administering an antibioticformulation to a patient in need thereof comprises inserting a tube intoa trachea of a patient. The method also comprises aerosolizing anantibiotic formulation to administer the antibiotic formulation to thepulmonary system of the patient. The antibiotic formulation consistsessentially of an anti-gram-negative antibiotic or salt thereof andwater.

In one or more embodiments, a method of administering an antibioticformulation to a patient in need thereof comprises aerosolizing anantibiotic formulation to administer the antibiotic formulation to thepulmonary system of the patient. The antibiotic formulation comprises ananti-gram-positive antibiotic or salt thereof at a concentration rangingfrom about 0.7 to about 0.9 of the water solubility limit, at 25° C. and1.0 atmosphere, of the anti-gram-positive antibiotic or salt thereof.

In one or more embodiments, a method of administering an antibioticformulation to a patient in need thereof comprises aerosolizing anantibiotic formulation using a vibrating mesh nebulizer, andadministering the antibiotic formulation to the pulmonary system of thepatient via an endotracheal tube, wherein the nebulizer is positioned inclose proximity to the endotracheal tube.

In one or more embodiments, a method of administering an antibioticformulation to a patient in need thereof comprises dissolving ananti-gram-positive antibiotic or salt thereof in a solvent to form anantibiotic formulation, wherein the anti-gram-positive antibiotic orsalt thereof is present at a concentration ranging from about 0.6 toabout 0.9 of the water solubility limit, at 25° C. and 1.0 atmosphere,of the anti-gram-positive antibiotic or salt thereof. The method alsoincludes aerosolizing the antibiotic formulation to administer theantibiotic formulation to the pulmonary system of the patient.

In one or more embodiments, a method of administering an antibioticformulation to a patient in need thereof comprises dissolving anantibiotic or salt thereof in a solvent to form an antibioticformulation. The method also includes aerosolizing the antibioticformulation to administer the antibiotic formulation to the pulmonarysystem of the patient, wherein the aerosolizing is conducted withinabout 16 hours of the dissolving.

In one or more embodiments, a method involves forming a powdercomprising an antibiotic or salt thereof. The method includes dissolvingan antibiotic or salt thereof in a solvent to form a solution having aconcentration ranging from about 60 mg/ml to about 120 mg/ml. The methodalso includes lyophilizing the solution to form the powder.

In one or more embodiments, a method involves forming a powdercomprising an antibiotic or salt thereof. The method comprisesdissolving an antibiotic or salt thereof in a solvent to form a solutionhaving a volume ranging from about 4.5 ml to about 5.5 ml. The methodalso includes lyophilizing the solution to form the dry powder.

Therefore, in one or more embodiments, the present invention involvesconcentrated antibiotic formulations. The antibiotic formulations maycomprise an aqueous composition of antibiotic or salt thereof beingpresent at a concentration ranging from about 0.6 to about 0.9, such asabout 0.7 to about 0.8, of the water solubility limit, at 25° C. and 1.0atmosphere, of the antibiotic or salt thereof.

The concentration of the antibiotic, corrected for potency, in one ormore embodiments, may range from about 40 mg/ml to about 200 mg/ml, suchas about 60 mg/ml to about 140 mg/ml, or about 80 mg/ml to about 120mg/ml. For example, in the case of anti-gram-negative antibiotics orsalts thereof, the concentration as corrected for potency may range fromabout 40 mg/ml to about 200 mg/ml, such as from about 90 mg/ml to about200 mg/ml, about 110 mg/ml to about 150 mg/ml, or about 120 mg/ml toabout 140 mg/ml. As another example, in the case of anti-gram-positiveantibiotics or salts thereof, the concentration as corrected for potencymay range from about 60 mg/ml to about 140 mg/ml, such as about 80 mg/mlto about 120 mg/ml.

The aqueous compositions typically have a pH that is compatible withphysiological administration, such as pulmonary administration. Forexample, the aqueous composition may have a pH ranging from about 3 toabout 7, such as about 4 to about 6.

In addition, the aqueous compositions typically have an osmolality thatis compatible with physiological administration, such as pulmonaryadministration. In one or more embodiments, the aqueous composition mayhave an osmolality ranging from about 90 mOsmol/kg to about 500mOsmol/kg, such as 120 mOsmol/kg to about 500 mOsmol/kg, or about 150mOsmol/kg to about 300 mOsmol/kg.

In one or more embodiments, the aqueous compositions are stable. Forinstance, in some cases, no precipitate forms in the aqueous compositionwhen the aqueous composition is stored for 1 year, or even 2 years, at25° C.

The potency of the antibiotic or salt thereof may range from about 500μg/mg to about 1100 μg/mg. In one or more embodiments, the potency ofanti-gram-negative antibiotics or salts thereof, such as gentamicin,typically ranges from about 500 μg/mg to about 1100 μg/mg, such as about600 μg/mg to about 1000 μg/mg, or about 700 μg/mg to about 800 μg/mg.The potency of anti-gram-positive antibiotics or salts thereof, such asvancomycin, typically ranges from about 900 μg/mg to about 1100 μg/mg,such as from about 950 μg/mg to about 1050 μg/mg.

The chromatographic purity level of the antibiotic or salt thereoftypically greater than about 80%, such as greater than about 85%,greater than about 90%, or greater than about 95%. In this regard, thereis generally no major impurity greater than about 10%, such as nogreater than about 5% or no greater than about 2%. For instance, theamount of heavy metals is typically less than about 0.005 wt %, such asless than about 0.004 wt %, less than about 0.003 wt %, less than about0.002 wt %, or less than about 0.001 wt %.

In the case of gentamicin, the compositions typically have a gentamicinC₁ content ranging from about 25% to about 50%, such as about 30% toabout 55%, about 35% to about 50%, or about 40% to about 45%, based onthe total amount of gentamicin. The compositions typically have agentamicin C_(1a) content ranging from about 10% to about 35%, such asabout 15% to about 30%, about 20% to about 25%, based on the totalamount of gentamicin. The compositions typically have a gentamicin C₂and C_(2a) content ranging from about 25 wt % to about 55 wt %, such asabout 30% to about 50%, about 30% to about 45%, or about 35% to about40%, based on the total amount of gentamicin.

In embodiments of the present invention comprising amikacin, thecompositions typically have an amikacin content ranging from about 25%to about 50%, such as about 30% to about 55%, about 35% to about 50%, orabout 40% to about 45%, based on the total amount of amikacin.

Nearly any anti-gram-negative, anti-gram-positive antibiotic, orcombinations thereof may be used. Additionally, antibiotics may comprisethose having broad spectrum effectiveness, or mixed spectrumeffectiveness. Antifungals, such as polyene materials, in particular,amphotericin B are also suitable for use herein. Examples ofanti-gram-negative antibiotics or salts thereof include, but are notlimited to, aminoglycosides or salts thereof. Examples ofaminoglycosides or salts thereof include gentamicin, amikacin,kanamycin, streptomycin, neomycin, netilmicin, paramecin, tobramycin,salts thereof, and combinations thereof. For instance, gentamicinsulfate is the sulfate salt, or a mixture of such salts, of theantibiotic substances produced by the growth of Micromonospora purpurea.Gentamicin sulfate, USP, may be obtained from Fujian FukangPharmaceutical Co., LTD, Fuzhou, China. Amikacin is typically suppliedas a sulfate salt, and can be obtained, for example, from Bristol-MyersSquibb. Amikacin may include related substances such as kanamicin.

Examples of anti-gram-positive antibiotics or salts thereof include, butare not limited to, macrolides or salts thereof. Examples of macrolidesor salts thereof include, but are not limited to, vancomycin,erythromycin, clarithromycin, azithromycin, salts thereof, andcombinations thereof. For instance, vancomycin hydrochloride is ahydrochloride salt of vancomycin, an antibiotic produced by certainstrains of Amycolatopsis orientalis, previously designated Streptomycesorientalis. Vancomycin hydrochloride is a mixture of related substancesconsisting principally of the monohydrochloride of vancomycin B. Likeall glycopeptide antibiotics, vancomycin hydrochloride contains acentral core heptapeptide. Vancomycin hydrochloride, USP, may beobtained from Alpharma, Copenhagen, Denmark.

In some embodiments, the composition comprises an antibiotic and one ormore additional active agents. The additional active agent describedherein includes an agent, drug, or compound, which provides somepharmacologic, often beneficial, effect. This includes foods, foodsupplements, nutrients, drugs, vaccines, vitamins, and other beneficialagents. As used herein, the terms further include any physiologically orpharmacologically active substance that produces a localized or systemiceffect in a patient. An active agent for incorporation in thepharmaceutical formulation described herein may be an inorganic or anorganic compound, including, without limitation, drugs which act on: theperipheral nerves, adrenergic receptors, cholinergic receptors, theskeletal muscles, the cardiovascular system, smooth muscles, the bloodcirculatory system, synoptic sites, neuroeffector junctional sites,endocrine and hormone systems, the immunological system, thereproductive system, the skeletal system, autacoid systems, thealimentary and excretory systems, the histamine system, and the centralnervous system.

Examples of additional active agents include, but are not limited to,anti-inflammatory agents, bronchodilators, and combinations thereof.

Examples of bronchodilators include, but are not limited to, β-agonists,anti-muscarinic agents, steroids, and combinations thereof. Forinstance, the steroid may comprise albuterol, such as albuterol sulfate.

Active agents may comprise, for example, hypnotics and sedatives,psychic energizers, tranquilizers, respiratory drugs, anticonvulsants,muscle relaxants, antiparkinson agents (dopamine antagonists),analgesics, anti-inflammatories, antianxiety drugs (anxiolytics),appetite suppressants, antimigraine agents, muscle contractants,additional anti-infectives (antivirals, antifungals, vaccines)antiarthritics, antimalarials, antiemetics, anepileptics, cytokines,growth factors, anti-cancer agents, antithrombotic agents,antihypertensives, cardiovascular drugs, antiarrhythmics, antioxicants,anti-asthma agents, hormonal agents including contraceptives,sympathomimetics, diuretics, lipid regulating agents, antiandrogenicagents, antiparasitics, anticoagulants, neoplastics, antineoplastics,hypoglycemics, nutritional agents and supplements, growth supplements,antienteritis agents, vaccines, antibodies, diagnostic agents, andcontrasting agents. The active agent, when administered by inhalation,may act locally or systemically.

The active agent may fall into one of a number of structural classes,including but not limited to small molecules, peptides, polypeptides,proteins, polysaccharides, steroids, proteins capable of elicitingphysiological effects, nucleotides, oligonucleotides, polynucleotides,fats, electrolytes, and the like.

Examples of active agents suitable for use in this invention include butare not limited to one or more of calcitonin, amphotericin B,erythropoietin (EPO), Factor VIII, Factor IX, ceredase, cerezyme,cyclosporin, granulocyte colony stimulating factor (GCSF),thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin,granulocyte macrophage colony stimulating factor (GMCSF), growthhormone, human growth hormone (HGH), growth hormone releasing hormone(GHRH), heparin, low molecular weight heparin (LMWH), interferon alpha,interferon beta, interferon gamma, interleukin-1 receptor,interleukin-2, interleukin-1 receptor antagonist, interleukin-3,interleukin-4, interleukin-6, luteinizing hormone releasing hormone(LHRH), factor IX, insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675, which isincorporated herein by reference in its entirety), amylin, C-peptide,somatostatin, somatostatin analogs including octreotide, vasopressin,follicle stimulating hormone (FSH), insulin-like growth factor (IGF),insulintropin, macrophage colony stimulating factor (M-CSF), nervegrowth factor (NGF), tissue growth factors, keratinocyte growth factor(KGF), glial growth factor (GGF), tumor necrosis factor (TNF),endothelial growth factors, parathyroid hormone (PTH), glucagon-likepeptide thymosin alpha 1, IIb/IIIa inhibitor, alpha-1 antitrypsin,phosphodiesterase (PDE) compounds, VLA-4 inhibitors, bisphosphonates,respiratory syncytial virus antibody, cystic fibrosis transmembraneregulator (CFTR) gene, deoxyribonuclease (Dnase),bactericidal/permeability increasing protein (BPI), anti-CMV antibody,13-cis retinoic acid, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin, teicoplanin, rampolanin, mideplanin,colistin, daptomycin, gramicidin, colistimethate, polymixins such aspolymixin B, capreomycin, bacitracin, penems; penicillins includingpenicillinase-sensitive agents like penicillin G, penicillin V,penicillinase-resistant agents like methicillin, oxacillin, cloxacillin,dicloxacillin, floxacillin, nafcillin; gram negative microorganismactive agents like ampicillin, amoxicillin, and hetacillin, cillin, andgalampicillin; antipseudomonal penicillins like carbenicillin,ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporinslike cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone,cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin,cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil,cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine,cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan,cefinetazole, ceftazidime, loracarbef, and moxalactam, monobactams likeaztreonam; and carbapenems such as imipenem, meropenem, pentamidineisethiouate, lidocaine, metaproterenol sulfate, beclomethasonediprepionate, triamcinolone acetamide, budesonide acetonide,fluticasone, ipratropium bromide, flunisolide, cromolyn sodium,ergotamine tartrate and where applicable, analogues, agonists,antagonists, inhibitors, and pharmaceutically acceptable salt forms ofthe above. In reference to peptides and proteins, the invention isintended to encompass synthetic, native, glycosylated, unglycosylated,pegylated forms, and biologically active fragments, derivatives, andanalogs thereof.

Active agents for use in the invention further include nucleic acids, asbare nucleic acid molecules, vectors, associated viral particles,plasmid DNA or RNA or other nucleic acid constructions of a typesuitable for transfection or transformation of cells, i.e., suitable forgene therapy including antisense. Further, an active agent may compriselive attenuated or killed viruses suitable for use as vaccines. Otheruseful drugs include those listed within the Physician's Desk Reference(most recent edition), which is incorporated herein by reference in itsentirety.

The amount of antibiotic or other active agent in the pharmaceuticalformulation will be that amount necessary to deliver a therapeuticallyor prophylactically effective amount of the active agent per unit doseto achieve the desired result. In practice, this will vary widelydepending upon the particular agent, its activity, the severity of thecondition to be treated, the patient population, dosing requirements,and the desired therapeutic effect. The composition will generallycontain anywhere from about 1 wt % to about 99 wt %, such as from about2 wt % to about 95 wt %, or from about 5 wt % to 85 wt %, of the activeagent, and will also depend upon the relative amounts of additivescontained in the composition. The compositions of the invention areparticularly useful for active agents that are delivered in doses offrom 0.001 mg/day to 100 mg/day, such as in doses from 0.01 mg/day to 75mg/day, or in doses from 0.10 mg/day to 50 mg/day. It is to beunderstood that more than one active agent may be incorporated into theformulations described herein and that the use of the term “agent” in noway excludes the use of two or more such agents.

Generally, the compositions are free of excessive excipients. In one ormore embodiments, the aqueous composition consists essentially of theanti-gram-negative antibiotic, such as amikacin, or gentamicin or both,and/or salts thereof and water.

Further, in one or more embodiments, the aqueous composition ispreservative-free. In this regard, the aqueous composition may bemethylparaben-free and/or propylparaben-free. Still further, the aqueouscomposition may be saline-free.

In one or more embodiments, the compositions comprise an anti-infectiveand an excipient. The compositions may comprise a pharmaceuticallyacceptable excipient or carrier which may be taken into the lungs withno significant adverse toxicological effects to the subject, andparticularly to the lungs of the subject. In addition to the activeagent, a pharmaceutical formulation may optionally include one or morepharmaceutical excipients which are suitable for pulmonaryadministration. These excipients, if present, are generally present inthe composition in amounts sufficient to perform their intendedfunction, such as stability, surface modification, enhancingeffectiveness or delivery of the composition or the like. Thus ifpresent, excipient may range from about 0.01 wt % to about 95 wt %, suchas from about 0.5 wt % to about 80 wt %, from about 1 wt % to about 60wt %. Preferably, such excipients will, in part, serve to furtherimprove the features of the active agent composition, for example byproviding more efficient and reproducible delivery of the active agentand/or facilitating manufacturing. One or more excipients may also beprovided to serve as bulking agents when it is desired to reduce theconcentration of active agent in the formulation.

For instance, the compositions may include one or more osmolalityadjuster, such as sodium chloride. For instance, sodium chloride may beadded to solutions of vancomycin hydrochloride to adjust the osmolalityof the solution. In one or more embodiments, an aqueous compositionconsists essentially of the anti-gram-positive antibiotic, such asvancomycin hydrochloride, the osmolality adjuster, and water.

Pharmaceutical excipients and additives useful in the presentpharmaceutical formulation include but are not limited to amino acids,peptides, proteins, non-biological polymers, biological polymers,carbohydrates, such as sugars, derivatized sugars such as alditols,aldonic acids, esterified sugars, and sugar polymers, which may bepresent singly or in combination.

Exemplary protein excipients include albumins such as human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,hemoglobin, and the like. Suitable amino acids (outside of thedileucyl-peptides of the invention), which may also function in abuffering capacity, include alanine, glycine, arginine, betaine,histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine,isoleucine, valine, methionine, phenylalanine, aspartame, tyrosine,tryptophan, and the like. Preferred are amino acids and polypeptidesthat function as dispersing agents. Amino acids falling into thiscategory include hydrophobic amino acids such as leucine, valine,isoleucine, tryptophan, alanine, methionine, phenylalanine, tyrosine,histidine, and proline.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like.

The pharmaceutical formulation may also comprise a buffer or a pHadjusting agent, typically a salt prepared from an organic acid or base.Representative buffers comprise organic acid salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid, or phthalic acid, Tris, tromethamine hydrochloride,or phosphate buffers.

The pharmaceutical formulation may also include polymericexcipients/additives, e.g., polyvinylpyrrolidones, celluloses andderivatized celluloses such as hydroxymethylcellulose,hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (apolymeric sugar), hydroxyethylstarch, dextrates (e.g., cyclodextrins,such as 2-hydroxypropyl-β-cyclodextrin andsulfobutylether-β-cyclodextrin), polyethylene glycols, and pectin.

The pharmaceutical formulation may further include flavoring agents,taste-masking agents, inorganic salts (for example sodium chloride),antimicrobial agents (for example benzalkonium chloride), sweeteners,antioxidants, antistatic agents, surfactants (for example polysorbatessuch as “TWEEN 20” and “TWEEN 80”), sorbitan esters, lipids (for examplephospholipids such as lecithin and other phosphatidylcholines,phosphatidylethanolamines), fatty acids and fatty esters, steroids (forexample cholesterol), and chelating agents (for example EDTA, zinc andother such suitable cations). Other pharmaceutical excipients and/oradditives suitable for use in the compositions according to theinvention are listed in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), bothof which are incorporated herein by reference in their entireties.

For MDI applications, the pharmaceutical formulation may also be treatedso that it has high stability. Several attempts have dealt withimproving suspension stability by increasing the solubility ofsurface-active agents in the HFA propellants. To this end U.S. Pat. No.5,118,494, WO 91/11173 and WO 92/00107 disclose the use of HFA solublefluorinated surfactants to improve suspension stability. Mixtures of HFApropellants with other perfluorinated cosolvents have also beendisclosed as in WO 91/04011. Other attempts at stabilization involvedthe inclusion of nonfluorinated surfactants. In this respect, U.S. Pat.No. 5,492,688 discloses that some hydrophilic surfactants (with ahydrophilic/lipophilic balance greater than or equal to 9.6) havesufficient solubility in HFAs to stabilize medicament suspensions.Increases in the solubility of conventional nonfluorinated MDIsurfactants (e.g. oleic acid, lecithin) can also reportedly be achievedwith the use of co-solvents such as alcohols, as set forth in U.S. Pat.Nos. 5,683,677 and 5,605,674, as well as in WO 95/17195. A particularlyuseful class of MDIs are those which use hydrofluoroalkane (HFA)propellants. The HFA propellants are further particularly well suited tobe used with stabilized dispersions of an active agent such asformulations and composition of aminoglycoside antibiotics. Suitablepropellants, formulations, dispersions, methods, devices and systemscomprise those disclosed in U.S. Pat. No. 6,309,623, the disclosure ofwhich is incorporated by reference in its entirety. All of theaforementioned references being incorporated herein by reference intheir entireties.

In one or more embodiments, the compositions comprise an aerosol havinga particle or droplet size selected to permit penetration into thealveoli of the lungs, such as a mass median aerodynamic diameter, lessthan about 10 μm, less than about 7.5 μm, less than about 5 μm, andusually being in the range of about 0.1 μm to about 5 μm.

The compositions of the present invention may be made by any of thevarious methods and techniques known and available to those skilled inthe art. In this regard, procedures such as lyophilizing antibiotics tomake powders and/or dissolving antibiotics in solvents are known in theart.

For instance, a solution of antibiotic, e.g., amikacin sulfate orgentamicin sulfate, may be made using the following procedure.Typically, manufacturing equipment is sterilized before use. A portionof the final volume, e.g., 70%, of solvent, e.g., water for injection,may be added into a suitable container. Antibiotic or salt thereof maythen be added. The antibiotic or salt thereof may be mixed untildissolved. Additional solvent may be added to make up the final batchvolume. The batch may be filtered, e.g., through a 0.2 μm filter into asterilized receiving vessel. Filling components may be sterilized beforeuse in filling the batch into vials, e.g., 10 ml vials.

As an example, the above-noted sterilizing may include the following. A5 liter type 1 glass bottle and lid may be placed in an autoclave bagand sterilized at elevated temperature, e.g., 121° C. for 15 minutes,using an autoclave. Similarly, vials may be placed into suitable racks,inserted into an autoclave bag, and sterilized at elevated temperature,e.g., 121° C. for 15 minutes, using an autoclave. Also similarly,stoppers may be placed in an autoclave bag and sterilized at elevatedtemperature, e.g., 121° C. for 15 minutes, using an autoclave. Beforesterilization, sterilizing filters may be attached to tubing, e.g., a 2mm length of 7 mm×13 mm silicone tubing. A filling line may be preparedby placed in an autoclave bag and sterilized at elevated temperature,e.g., 121° C. for 15 minutes, using an autoclave.

The above-noted filtration may involve filtration into a laminar flowwork area. The receiving bottle and filters may be set up in the laminarflow work area.

The above-noted filling may also be conducted under laminar flowprotection. The filling line may be unwrapped and placed into thereceiving bottle. The sterilized vials and stoppers may be unwrappedunder laminar flow protection. Each vial may be filled, e.g., to atarget fill of 5.940 g, and stoppered. A flip off collar may be appliedto each vial. The sealed vials may be inspected for vial leakage,correct overseals, and cracks.

As another example, one or more antibiotics, e.g., vancomycin,gentamicin or amikacin, and/or a salt thereof, may be prepared bylyophilizing the antibiotic to form a powder for storage. The powder isthen reconstituted prior to use. This technique may be used when theantibiotic is unstable in solution.

In one or more embodiments, the powder making process may begin withforming a solution to be lyophilized. For example, an antibiotic or saltthereof, such as amikacin, gentamicin or vancomycin and/or saltsthereof, may be dissolved in a solvent to form a solution having anantibiotic concentration ranging from about 80 mg/ml to about 150 mg/ml,such as about 90 mg/ml to about 130 mg/ml, or about 100 mg/ml to about124 mg/ml. The solution to be lyophilized may have a volume ranging fromabout 4.5 ml to about 5.5 ml, such as about 5 ml.

In other embodiments, the powder making process may begin with forming asolution of an anti-gram negative antibiotic or salt thereof, such asamikacin or salt thereof. The antibiotic and/or salt may be dissolved ina solvent to form a solution having a concentration ranging from about80 mg/ml to about 130 mg/ml, such as about 90 mg/ml to about 120 mg/ml,or about 100 mg/ml to about 110 mg/ml. The solution to be lyophilizedmay have a volume ranging from about 4.5 ml to about 5.5 ml, such asabout 5 ml.

The solvent for the solution to be lyophilized may comprise water. Thesolution may be excipient-free. For instance, the solution may becryoprotectant-free.

In one or more embodiments, a suitable amount (e.g., 120 g per liter offinal solution) of drug substance (for example vancomycin hydrochloride)may be dissolved, e.g., in about the 75% of the theoretical total amountof water for injection under nitrogen bubbling. The dissolution time maybe recorded and appearance may be evaluated.

Then, the dilution to the final volume with WFI may be carried out.Final volume may be checked. Density, pH, endotoxin, bioburden, andcontent by UV may be measured both before and after sterile filtration.

The solution may be filtered before lyophilizing. For instance, a double0.22 μm filtration may be performed before filling. The filters may betested for integrity and bubble point before and after the filtration.

Pre-washed and autoclaved vials may be aseptically filled using anautomatic filling line to a target of 5 ml per vial and then partiallystoppered. In process check for fill volumes may be done by checking thefill weight every 15 minutes.

The lyophilizing is generally conducted within about 72 hours, such aswithin about 8 hours, or within about 4 hours, of the dissolving.

In one or more embodiments, the lyophilizing comprises freezing thesolution to form a frozen solution. The frozen solution is typicallyheld at an initial temperature ranging from about −40° C. to about −50°C., such as about −45° C. During the initial temperature period, thepressure around the frozen solution is typically atmospheric pressure.The initial temperature period typically ranges from about 1 hour toabout 4 hours, such about 1.5 hours to about 3 hours, or about 2 hours.

The lyophilizing may further comprise raising a temperature of thefrozen solution to a first predetermined temperature, which may rangefrom about 10° C. to about 20° C., such as about 15° C. The time for theheat ramp from the initial temperature to the first predeterminedtemperature generally ranges from about 6 hours to about 10 hours, suchas about 7 hours to about 9 hours.

During the first predetermined temperature period, the pressure aroundthe solution typically ranges from about 100 μbar to about 250 μbar,such as about 150 μbar to about 225 μbar. The solution may be held atthe first predetermined temperature for a period ranging from about 20hours to about 30 hours, such as from about 24 hours.

The lyophilizing may still further comprise raising a temperature of thesolution to a second predetermined temperature, which may range fromabout 25° C. to about 35° C., such as about 30° C. During the secondpredetermined temperature period, the pressure around the frozensolution typically ranges from about 100 μbar to about 250 μbar, such asabout 150 μbar to about 225 μbar. The solution may be held at the secondpredetermined temperature for a period ranging from about 10 hours toabout 20 hours.

In view of the above, the lyophilization cycle may comprise a freezingramp, e.g., from 20° C. to −45° C. in 65 minutes, followed by a freezesoak, e.g., at −45° C. for 2 hours. Primary drying may be accomplishedwith a heating ramp, e.g., from −45° C. to 15° C. in 8 hours, followedby a temperature hold, e.g., at 15° C. for 24 hours at a pressure of 200μbar. Secondary drying may be accomplished with a heating ramp, e.g.,from 15° C. to 30° C. in 15 minutes, followed by a temperature hold at30° C. for 15 hours at a pressure of 200 μbar. At the end of thelyophilization cycle, the vacuum may be broken with sterile nitrogen,and the vials may be automatically stoppered.

The water content of the powder e.g., vancomycin powder, or amikacinpowder, is typically less than about 7 wt %, such as less than about 5wt %, less than about 4 wt %, less than about 3 wt %, or less than about2 or 1 wt %.

The chromatographic purity level of the powder, e.g., vancomycin powder,or amikacin powder, typically greater than about 80%, such as greaterthan about 90%, greater than about 95%, or greater than about 97%. Inthis regard, there is generally no major impurity greater than about10%, such as no greater than about 7% or no greater than about 5%. Forinstance, the amount of heavy metals is typically less than about 0.005wt %, such as less than about 0.004 wt %, less than about 0.003 wt %,less than about 0.002 wt %, or less than about 0.001 wt %.

The powder is capable of being reconstituted with water at 25° C. and1.0 atmosphere and with manual agitation, in less than about 60 seconds,such as less than about 30 seconds, less than about 15 seconds, or lessthan about 10 seconds.

The powder typically has a large specific surface area that facilitatesreconstitution. The specific surface area typically ranges from about 5m²/g to about 20 m²/g, such as about 8 m²/g to 15 m²/g, or about 10 m²/gto 12 m²/g.

Upon reconstitution with water, the antibiotic solution (such asvancomycin or amikacin) typically has a pH that ranges from about 2.5 toabout 7, such as about 3 to about 6. Amikacin in particular may have apH of about 5.5 to about 6.3.

In addition to use formulations for nebulization, the formulations ofthe present invention may be administered other routes, e.g., parenteraladministration.

One or more embodiments involve methods for treating or preventingpulmonary infections, including nosocomial infections, in animals,including, especially, humans. The method generally comprisesadministering to an animal subject or human patient in need thereof, asan aerosol, a therapeutically or prophylactically effective amount ofthe antibiotic or salt thereof. Several antibiotics may be delivered incombination according to the invention, or in seriatim. In one or moreembodiments, the amounts delivered to the airways, if deliveredsystemically in such amounts, would not be sufficient to betherapeutically effective and would certainly not be enough to inducetoxicity. At the same time, in such embodiments, such amounts can resultin sputum levels of antibiotic of more than about 10-100 times theminimum inhibitory concentration (“MIC”).

In one particular embodiment, the pharmaceutical formulation comprisesan antibiotic for administration to a ventilated patient to treat orprevent ventilator associated pneumonia (VAP) and/or hospital-acquiredpneumonia (HAP) and/or community acquired pneumonia (CAP) as well asother forms of pneumonia, and other respiratory infections orconditions. Such administration is described in U.S. patent applicationSer. Nos. 10/430,658; 10/430,765; and 10/991,092, and in U.S.Provisional Application Nos. 60/378,475; 60/380,783; 60/420,429;60/439,894; 60/442,785; 60/682,099, and in U.S. Patent ApplicationPublication No. 2005/021766, all of which are incorporated herein byreference in their entireties.

In one aspect, the aerosolized particles are prevented from undergoingsignificant hygroscopic enlargement, since particles enrobed in waterwill tend to condense on the walls. For instance, the method may involvereducing humidity in the ventilator circuit by a predetermined amountbefore nebulization begins. In this embodiment, the humidity mayfacilitate an MMAD of less than about 3 μm or less than about 1.5 μm. Inanother embodiment, each aerosol particle is delivered enrobed in asubstantially anhygroscopic envelope.

Of course, embodiments can be used where diameters are greater.Moreover, in some cases, the present invention contemplates adjustmentsto the surface electrical charges on the particles or the walls. Forexample, assuming surface charge on the device is important, the presentinvention contemplates embodiments wherein the components of the deviceconnectors are made of metal (or at least coated with metal).Alternatively, the components can be treated with agents (e.g. wettingagents, detergents, soaps) to adjust surface charge.

In one aspect, the method comprises inserting an aerosol delivery end ofthe device within said patient's trachea to create a positioned device.The antibiotic composition is aerosolized under conditions such that thecomposition is delivered through said aerosol delivery end of the deviceto the patient, wherein the aerosol first contacts the patient's trachea(thereby bypassing the oro-pharynx). The method may involveadministering a mixture of antibiotics and is particularly appropriatefor intubated patients.

In another aspect, a method of administering comprises administering tofree breathing patients by way of an aerosol generator device and/orsystem for administration of aerosolized medicaments such as thosedisclosed in U.S. Patent Application Publication Nos. 20050235987,20050211253, 20050211245, 20040035413, and 20040011358, the disclosuresof which are incorporated herein by reference in their entireties.

Such devices may deliver medicament phasically or non-phasically.Additionally or alternatively, such devices may incorporate a chamber orreservoir to accumulate and periodically dispense the aerosolizedmedicament. In one or more embodiments, an aerosolized medicamentcomprises amikacin.

In one or more embodiments, the method of administering an antibioticformulation involves dissolving an antibiotic or salt thereof in asolvent to form an antibiotic formulation. The aerosolizing is conductedwithin about 16 hours, such as with about 12 hours, or within about 8hours, of the dissolving.

In another aspect, particular with respect to “constant-flow”ventilators, the present invention contemplates limiting the deliveryevent to the inspiratory phase of the ventilator cycle and, if possible,at a reduced flow-rate. Thus, in one embodiment, aerosolization isactuated during (or in fixed relation to) the inspiration phase of thebreathing cycle.

It is not intended that the present invention be limited to particulardosages. On the other hand, the efficiency of the aerosol systems andmethods described herein permit amounts to be delivered that are too lowto be generally effective if administered systemically, but arenonetheless effective amounts when administered in a suitable andpharmaceutically acceptable formulation directly to the airway.Importantly, while efficiencies can be increased, in some embodimentsefficiencies are not increased at the expense of control over the dose.Thus, lower efficiencies are contemplated as preferred when delivery ismore reproducible.

It is not intended that the present invention be limited toantimicrobials that only kill particular organisms. The presentinvention contemplates drugs and drug combinations that will address awide variety of organisms. In one or more embodiments, the presentinvention contemplates drugs or drug combinations effective in thetreatment of infections caused by P. aeruginosa, S. aureus, H.influenza, and S. pneumoniae and/or antibiotic-resistant strains ofbacteria such as methicillin-resistant S. aureus, and Acetinobacterspecies, among others.

Moreover, while certain embodiments of the present invention arepresented in the context of the intubated patient, other patients atrisk for infection are contemplated as treatable with the compositions,methods, and devices of the present invention. For example, the elderly(particularly those in nursing homes), horses, dogs and cats incompetitions (show and racing animals), animals that frequently travel(e.g., circus animals), animals in close quarters (e.g., zoos or farms),humans and animals in general are at risk for lung infections. Thepresent invention contemplates delivery of aerosols to the tracheaand/or deep lung for such individuals—both prophylactically (i.e.,before symptoms) and under acute conditions (i.e., aftersymptoms)—wherein said aerosols comprise antimicrobials, and inparticular, the antibiotic mixtures described above.

In one embodiment, the present invention contemplates administering theappropriate medication to a patient diagnosed with ARDS or chronicobstructive pulmonary disease (COPD).

One or more embodiments are directed to unit doses comprising acontainer and the compositions.

Examples of the container include, but are not limited to, vials,syringes, ampoules, and blow fill seal. For instance, the vial may be acolorless Type I borosilicate glass ISO 6R 10 mL vial with a chlorobutylrubber siliconized stopper, and rip-off type aluminum cap with coloredplastic cover.

The amount of the composition in the unit dose typically ranges fromabout 2 ml to about 15 ml, such as from about 3 ml to about 10 ml, about4 ml to about 8 ml, or about 5 ml to about 6 ml.

The amount of the antibiotic in the unit dose, adjusted for potency,typically ranges from about 150 mg to about 900 mg, such as about 400 mgto about 750 mg. For instance, an amount of the anti-gram-negativeantibiotic or salt thereof may range from about 400 mg to about 750 mg.As another example, the amount of anti-gram-positive antibiotic or saltthereof may range from about 150 mg to about 450 mg, or from about 550mg to about 900 mg.

One or more embodiments are directed to kits. For instance, the kit mayincludes a first container containing a first aqueous solutioncomprising anti-gram-negative antibiotic or salt thereof and a secondcontainer containing a second aqueous solution comprisinganti-gram-negative antibiotic or salt thereof. A concentration, or anamount, or both, of the first aqueous solution is different from aconcentration, or an amount, or both, of the second aqueous solution.For instance, the amount of the first aqueous solution may range fromabout 2 ml to about 5 ml, and the amount of the second aqueous solutionmay range from about 5 ml to about 8 ml.

In one or more embodiments, the kit includes a first containercontaining a first aqueous solution comprising anti-gram-negativeantibiotic or salt thereof. A second container contains a second aqueoussolution comprising anti-gram-positive antibiotic or salt thereof. Theconcentrations and/or amounts of the anti-gram-negative antibiotic orsalt and the anti-gram-positive antibiotic or salt may be the same ordifferent.

In one or more embodiments, a kit includes a first container containinga first composition comprising an antibiotic or salt thereof. A secondcontainer contains a second composition comprising water. The firstcomposition and/or the second composition comprises an osmolalityadjuster.

In one or more embodiments, a kit includes a first container containinga powder comprising anti-gram-positive antibiotic or salt thereof. Asecond container contains a powder comprising anti-gram-positiveantibiotic or salt thereof. A concentration, or an amount, or both ofthe anti-gram-positive antibiotic or salt thereof in the first containeris different from a concentration, or an amount, or both of theanti-gram-positive antibiotic or salt thereof in the second container.

For instance, the amount of the anti-gram-positive antibiotic or saltthereof in the first container may range from about 400 mg to 600 mg.The amount of the anti-gram-positive antibiotic or salt thereof in thesecond container may range from about 600 mg to about 800 mg.

In another aspect, a kit may include a first container containing asolution comprising anti-gram-negative antibiotic or salt thereof. Asecond container may contain a powder comprising anti-gram-positiveantibiotic or salt thereof. Alternatively, the anti-gram-negativeantibiotic or salt thereof may be a powder, and the anti-gram-positiveantibiotic or salt thereof may be a solution or dispersion. An amount ofthe anti-gram-positive antibiotic or salt thereof generally ranges fromabout 150 mg to about 900 mg.

The kits may further comprise a package, such as a bag, that containsthe first container and the second container.

The kits may further comprise an aerosolization apparatus. Theaerosolization apparatus may be of any type that is capable of producingrespirable particles or droplets. Alternatively, the antibiotic may bedissolved in or suspended in a liquid propellant, as described in U.S.Pat. No. 5,225,183; 5,681,545; 5,683,677; 5,474,759; 5,508,023;6,309,623; or 5,655,520, all of which are incorporated herein byreference in their entireties. In such cases, the aerosolizationapparatus may comprise a metered dose inhaler (MDI).

Alternatively or additionally, the pharmaceutical formulation may be ina liquid form and may be aerosolized using a nebulizer as described inWO 2004/071368, which is herein incorporated by reference in itsentirety, as well as U.S. Published Application Nos. 2004/0011358 and2004/0035413, which are both herein incorporated by reference in theirentireties. Other examples of nebulizers include, but are not limitedto, the Aeroneb®Go or Aeroneb®Pro nebulizers, available from Aerogen,Inc. of Mountain View, Calif.; the PARI eFlow and other PARI nebulizersavailable from PARI Respiratory Equipment, Inc. of Midlothian, Va.; theLumiscope® Nebulizer 6600 or 6610 available from Lumiscope Company, Inc.of East Brunswick, N.J.; and the Omron NE-U22 available from OmronHealthcare, Inc. of Kyoto, Japan.

It has been found that a nebulizer of the vibrating mesh type, such asone that that forms droplets without the use of compressed gas, such asthe Aeroneb® Pro provides unexpected improvement in dosing efficiencyand consistency. By generating fine droplets by using a vibratingperforated or unperforated membrane, rather than by introducingcompressed air, the aerosolized pharmaceutical formulation can beintroduced into the ventilator circuit without substantially affectingthe flow characteristics within the circuit and without requiring asubstantial re-selection of the ventilator settings. In addition, thegenerated droplets when using a nebulizer of this type are introduced ata low velocity, thereby decreasing the likelihood of the droplets beingdriven to an undesired region of the ventilator circuit. Furthermore,the combination of a droplet forming nebulizer and an aerosol introduceras described is beneficial in that there is a reduction in thevariability of dosing when the ventilator uses different tidal volumes,thus making the system more universal.

Using an adaptor, device or system as disclosed in U.S. application Ser.No. 10/991,092 and/or U.S. Provisional Application No. 60/682,099,and/or U.S. Application Publication No. 2005/0217666, all of which areincorporated herein by reference in their entireties, in connection withthe administration of aerosolized antibiotics offers substantialbenefits. For example, when using such adaptors, substantially lesspharmaceutical formulation is lost to the environment which results in areduction in bacterial resistance against the antibiotic. In addition,the adaptors, devices or systems are able to deliver a more consistentdose which is particularly useful for antibiotic therapy.

FIG. 1A shows an embodiment of an adapter or system for aerosol deliveryof medicaments, comprising a pulmonary drug delivery system (“PDDS”) 100suitable for use with the present invention. The PDDS 100 may include anebulizer 102 (also called an aerosolizer), which aerosolizes a liquidmedicament stored in reservoir 104. The aerosol exiting nebulizer 102may first enter the T-adaptor 106 that couples the nebulizer 102 to theventilator circuit. The T-adaptor 106 is also coupled to the circuit wye108 that has branching ventilator limbs 110 and 112.

Coupled to one of the ventilator limbs 110 or 112 may be an air pressurefeedback unit 114, which equalizes the pressure in the limb with the airpressure feedback tubing 116 connected to the control module 118. In theembodiment shown, feedback unit 114 has a female connection end (e.g.,an ISO 22 mm female fitting) operable to receive ventilator limb 112,and a male connection end (e.g., an ISO 22 mm male fitting) facingopposite, and operable to be inserted into the ventilator. The feedbackunit may also be operable to receive a filter 115 that can trapparticulates and bacteria attempting to travel between the ventilatorcircuit and tubing 116.

The control module 118 may monitor the pressure in the ventilator limbvia tubing 116, and use the information to control the nebulizer 102through system cable 120. In other embodiments (not shown) the controlmodule 118 may control aerosol generation by transmitting wirelesssignals to a wireless control module on the nebulizer 102.

During the inhalation phase of the patient's breathing cycle,aerosolized medicament entering T-adaptor 106 may be mixed with therespiratory gases from the inspiratory ventilator limb 112 flowing tothe patient's nose and/or lungs. In the embodiment shown, the aerosoland respiratory gases flow through nose piece 122 and into the nasalpassages of the patient's respiratory tract.

Other embodiments of the circuit wye 108 shown in FIG. 1A are alsocontemplated in embodiments of the invention.

Referring to FIG. 1B, a nebulizer 85, which may have a top portion 93through which liquid may be provided may be incorporated into aventilator breathing circuit of a ventilated patient. The breathingcircuit may comprise a “Y” connector 88, which may in turn have an inletportion 89, an endotracheal tube portion 90 and an outlet portion 91.The inlet portion 89 carries air provided from the ventilator 92 towardthe patient. The endotracheal tube portion 90 of the Y connector 88carries the incoming air to the patient's respiratory tract; thisdirection is represented by arrow “a”. The endotracheal tube portion 90also carries the patient's exhalation to the outlet portion 91 of the Yconnector 88, and the outlet portion may lead to an exhaust, representedby arrow “b”, to remove the patient's exhalation from the system. Thenebulizer 85 of the present invention aerosolization element generatesan aerosol cloud 94 that remains substantially within the inlet portion89 of the Y connector 88 when there is no inspiratory air flowingthrough the inlet portion, by virtue of the aerosolization element, asdescribed above, producing a low velocity mist. In this manner, aerosolthat is generated when there is no inhalation air being provided willnot be carried out through the outlet portion 91 of the Y connector andlost to the ambient environment. Accordingly, a dose of aerosolizedmedication may be preloaded, i.e., produced and placed substantiallywithin the inlet portion 89 prior to an inhalation phase being sent bythe ventilator 92. In this manner, such medication can be swept into apatient's respiratory system at the very start of the inhalation cycle.This may be of particular benefit in the case of neonatal patients andin other instances in which only the initial blast of inhalation phasewill reach the target portion of the respiratory system. In alternateembodiments, the ventilator may generate a continuous bias flow of gasthrough the ventilator circuit. The bias flow may push some of theaerosolized medicament through the outlet portion 91, but there is stillan overall benefit from having the aerosolized medicament preloadedthrough the ventilator circuit.

Referring now to FIG. 2A, an embodiment of an off-ventilatorconfiguration of an adapter and/or system for pulmonary delivery isshown. In FIG. 2A, the adapter 400 is intended for off-ventilator use,and includes an endpiece 402 that is coupled to a nebulizer 404 and wye406. The nebulizer 404 may include reservoir 408, which supplies theliquid medicament that is aerosolized into connector 410. The connector410 can provide a conduit for the aerosolized medicament and gases totravel from the wye 406 to endpiece 402, and then into the patient'smouth and/or nose. The first wye limb 412 may be connected to a pump orsource of pressurized respiratory gases (not shown), which flow throughthe wye limb 412 to the endpiece 402. A one-way valve 413 may also beplaced in the limb 412 to prevent respired gases from flowing back intothe pump or gas source. The limb 412 may also include a pressurefeedback port 414 that may be connected to a gas pressure feedback unit(not shown). In the embodiment shown, a feedback filter 416 may becoupled between the port 414 and feedback unit.

The off-ventilator adapter 400 may also include a second wye limb 420,which includes a filter 422 and one-way valve 424, through which gasesmay pass during an exhalation cycle. The filter 422 may filter outaerosolized medicament and infectious agents exhaled by the patient toprevent these materials from escaping into the surrounding atmosphere.The one-way valve 424 can prevent ambient air from flowing back into theadapter 400.

A general form of an aerosolized composition delivery system 1100 isshown in FIG. 2B. The aerosolized composition delivery system 1100delivers an aerosolized composition to a portion of a user's respiratorytract, such as the user's lungs. The aerosolized composition deliverysystem 1100 is useful in delivering the aerosolized composition to apatient whose breathing is being assisted by a ventilator 1105 but mayalso be configured to be used to deliver a composition to anon-ventilated patient. The ventilator circuit 1110 is showndiagrammatically in FIG. 2B. Extending from the ventilator 1105 is aninhalation line 1115 and an exhalation line 1120. The inhalation line1115 and the exhalation line 1120 are both composed of tubing having anairflow lumen extending therethrough. The inhalation line 1115 and theexhalation line 1120 meet at an adaptor 1145 remote from the ventilator1105. At the adapter 1145 the lumen of the inhalation line 1115 is incommunication with the lumen from the exhalation line 1120, and bothlumens are in communication with a patient line 1130. The patient line1130 comprises a lumen that extends to the lumen of an endotracheal ortracheostomy tube 1135, which is inserted into a patient. The tube 1135has an opposite end that may extend into or near the lungs of the user.Accordingly, in use, oxygenated air is introduced into the inhalationline 1115 by the ventilator 1105. The oxygenated air passes through thelumen of the inhalation line 1115, into the patient line 1130, throughthe lumen of the tube 1135, and into the lungs of the patient. Thepatient then exhales, either naturally or by applying negative pressurefrom the ventilator, and the exhaled air passes through the tube 1135,through the patient line 1130, and through the exhalation line 1120 tothe ventilator 1105. The cycle is continuously repeated to assist thepatient's breathing or to entirely control the breathing of the patient.

The adapter 1145 introduces aerosolized composition into the ventilatorcircuit 1110. The aerosol that is introduced by the adapter 1145 isgenerated by an aerosolization apparatus 1150, which comprises areservoir for containing a composition. Thus, in one or moreembodiments, aerosolization energy is supplied to the aerosolizationdevice by an energy source 1160 to generate the aerosolized composition.The aerosolized pharmaceutical formulation passes through a passage 1165to the adapter 1145 where it may be introduced into the ventilatorcircuit 1110. The aerosolization apparatus 1150 may be, for example, ajet nebulizer where the energy source is compressed air, a vibratingmesh nebulizer where the energy source is wave energy, an ultrasonicnebulizer, or a metered dose inhaler where the energy source is apropellant that boils under ambient conditions.

Examples of the adaptor 1145 for introducing the aerosolizedpharmaceutical formulation are disclosed in U.S. application Ser. No.10/991,092, filed Nov. 17, 2004, and U.S. Provisional Application No.60/682,099, which applications are herein incorporated by reference intheir entirety.

The introduction of the aerosolized pharmaceutical formulation at theadapter 1145 is advantageous in many respects over systems where theaerosol is introduced into the inhalation line 1115 or within theventilator 1105. For example, by introducing the aerosolizedpharmaceutical formulation at the adapter 1145, the ventilator circuitvolume from the point of introduction to the patient's lungs issubstantially reduced. Accordingly, the aerosolized pharmaceuticalformulation is more concentrated and is less diffused throughout theventilator circuit 1110. In addition, if the formulation is added in theinhalation line 1115, much of the formulation is drawn into theexhalation line 1120, further limiting the efficiency of theadministration. Because of this diffusion and reduced efficiency, theconsistency of dosing is difficult to control in known systems. Also,the presence of high quantities of the aerosolized pharmaceuticalformulation that are not administered to the lungs of the patient may beundesirable in that much of the aerosol may be introduced into theenvironment where it may be inhaled by healthcare workers or others.

Therefore, the adaptor 1145 of the invention has been designed tointroduce the aerosolized pharmaceutical formulation in an improvedmanner to increase the efficiency and/or the consistency of the dosing.The adaptor 1145 serves to reduce the amount of aerosolizedpharmaceutical formulation that is drawn into the exhalation line 1120of the ventilator circuit 1120.

The adaptors of the present invention when used in a ventilator circuitare often able to reproducibly and efficiently deliver pharmaceuticalformulation. For instance, the present invention is typically able toreproduce the delivered dose within about ±10%, ±8%, ±6%, ±4%, ±2%, or±1%, of the total nominal dose. The present invention is often able toachieve a delivered efficiency of at least about 30%, such as at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, or at least about 90%.

The adaptor of the present invention typically has minimal impact on thepatient to ventilator interface. The minimal impact allows theventilator to react more efficiently to the patient. The adaptor andvalves are arranged so that at an air flow rate of 60 L/min, thepressure drop between the first end and the second end of the adaptor isoften less than about 50 cm H₂O, such as less than about 30 cm H₂O, lessthan about 5 cm H₂O, less than about 4 cm H₂O, less than about 3 cm H₂O,less than about 2H₂O, less than about 1 cm H₂O, less than about 0.5 cmH₂O, or less than about 0.1 cm H₂O, and may range from about 0.05 cm H₂Oto about 10 cm H₂O, about 1 cm H₂O to about 5 cm H₂O, or about 2 cm H₂Oto about 4 cm H₂O. At an air flow rate of 30 L/min, the pressure dropbetween the first end and the second end of the adaptor is typicallyranges from about 1 cm H₂O to about 2 cm H₂O.

The adaptor may be made of a transparent, translucent, or opaquematerial. Using a transparent material is advantageous because the usercan visually inspect the functioning of the adaptor. Examples ofmaterials for the adaptor include, but are not limited, to polymers,such as polypropylene, SAN (styrene acrylonitrile copolymer), ABS(acrylonitrile-butadiene-styrene), polycarbonate, acrylic polysulfone,K-Resin® styrene-butadiene-copolymer (available from Chevron PhillipsChemical), polyethylene, PVC (polyvinyl chloride), polystyrene, and thelike.

For vibrating mesh nebulizers, such as the Aeroneb Pro and the PARIeFlow, reproducible administrations can result from smaller firstchannel volumes. It has been determined, for example, that the firstchannel volume for an adaptor 1145 used with a vibrating mesh nebulizermay be any volume greater than about 10 ml, such as from about 10 ml toabout 1000 ml, about 50 ml to about 200 ml, or about 90 ml. Both thestored volume and valving affect the performance of the presentinvention.

Additional examples of devices and methods are disclosed in U.S. patentapplication Ser. No. 11/436,329, “Valves, Devices, and Methods forEndobronchial Therapy,” filed May 18, 2006, which is incorporated hereinby reference in its entirety.

The present invention is not limited to any precise desired outcome whenusing the above-described compositions, devices, and methods. However,it is believed that the compositions, devices, and methods of thepresent invention may result in a reduction in mortality rates ofintubated patients, a decrease in the incidence of resistance (or atleast no increase in resistance) because of the reduced systemicantibiotic exposure and elevated exposure at the targeted mucosalsurface of the lung caused by local administration. As noted above, itis contemplated that the compositions, devices, and methods of thepresent invention are useful in the treatment of pneumonia (and may bemore effective than systemic treatment—or at the very least, a usefuladjunct). It is believed that related infections may also be preventedor reduced (e.g., prevention of sepsis, suppression of urinary tractinfections, etc.)

Of course, a reduced use of systemic antibiotics because of the efficacyof the compositions, devices, and methods of the present invention mayresult in reduced cost, reduced time on IV lines, and/or reduced time oncentral lines). Moreover, such a reduction should reduce antibiotictoxicity (as measured by reduced incidence of diarrhea and C. difficileinfection, better nutrition, etc.)

It is believed that the compositions, devices, and methods of thepresent invention will locally result in a reduction of the ET/Trachtube biofilm. This should, in turn, get rid of secretions, decreaseairway resistance, and/or decrease the work of breathing. The lattershould ease the process of weaning the patient off of the ventilator.

The present invention contemplates specific embodiments that can replacecommonly used elements of a ventilator system. In one or moreembodiments, the present invention contemplates an adapter attachable toa ventilator circuit and to an endotracheal tube, wherein the adaptorcomprises an aerosol generator. While not limited to any precise desiredoutcome, it is contemplated that the adapter with integral generatorwill reduce the effects of the ventilator on all conventional aerosolsystems (jet, ultrasonic and MDI), and at the same time enhance thepositive qualities of a device like the Aerogen™ pro. Again, while notlimited to any precise desired outcome, it is contemplated that theadapter with integral generator will (1) reduce variability in delivery(reduced effects of humidification, bias flow, continuous vsbreath-actuated) so as to achieve the same delivery (no matter whatcommercial ventilator system is used); (2) allow for maximal effects ofbreath actuation; and (3) allow for maximal effect to enhanced nebulizerefficiency using nebulizers having no dead volume.

The present invention is not limited to the precise configuration ornature of the circuit. In one embodiment, said circuit is a closedcircuit. In another embodiment, said circuit is an open circuit.

Again, the present invention is not limited to particular ventconfigurations. In one embodiment, said inspiratory and said expiratorylines are connected to a mechanical ventilator. In one embodiment, saidmechanical ventilator controls a breathing cycle, said cycle comprisingan inspiration phase. In one embodiment, the aerosol is administeredduring the inspiration phase of the breathing cycle.

Although the present invention has been described in considerable detailwith regard to certain versions thereof, other versions are possible,and alterations, permutations and equivalents of the version shown willbecome apparent to those skilled in the art upon a reading of thespecification and study of the drawings. For example, the relativepositions of the elements in the aerosolization device may be changed,and flexible parts may be replaced by more rigid parts that are hinged,or otherwise movable, to mimic the action of the flexible part. Inaddition, the passageways need not necessarily be substantially linear,as shown in the drawings, but may be curved or angled, for example.Also, the various features of the versions herein can be combined invarious ways to provide additional versions of the present invention.Furthermore, certain terminology has been used for the purposes ofdescriptive clarity, and not to limit the present invention. Therefore,any appended claims should not be limited to the description of thepreferred versions contained herein and should include all suchalterations, permutations, and equivalents as fall within the truespirit and scope of the present invention.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples, are, however, merelyrepresentative of methods of practicing one or more embodiments of thepresent invention and should not be read as limiting the scope of theinvention.

Example 1

This Example involves determining the solubility of gentamicin sulfatein water and saline. The required strengths were initially set at 20mg/ml, 40 mg/ml, and up to 200 mg/ml.

Water Solubility Determinations

Solubility in water was determined via visual assessment. Osmolality andpH were also determined.

The batch size of all the solutions manufactured for the solubilitydetermination studies was 10 ml. The method of manufacture consisted ofweighing the appropriate amount of gentamicin sulfate and then taking tofinal volume with water. It was noted that especially for higherconcentrations, the solution was first shaken by hand and then placed ona magnetic stirrer to ensure complete dissolution.

Table 1 lists the pH and osmolality values obtained for solutions ofgentamicin sulfate in water for injection (WFI) with concentrationsranging from 20 mg/ml to 400 mg/ml.

TABLE 1 Test Matrix for Gentamicin Solution in WFI Active ConcentrationWeight of Gentamicin Sulfate Osmolality (mg/ml) Dispensed⁽*⁾ (mg/ml) pH(mOsmol/kg) 20 34 4.81 61 40 68 4.72 101 80 136 4.92 197 120 204 4.93275 200 340 5.01 524 250 425 5.06 1178 300 510 5.13 2013 350 595 5.20 NR400 680 5.26 NR ⁽*⁾Activity of gentamicin sulfate = 58.8%, so conversionfactor = 1.701 NR: No result, sample did not freeze

As seen in Table 1, all the solutions had a pH that was higher than 4,which is considered to be acceptable for drug delivery to the lungs.However, with regard to osmolality readings, doses greater than 200mg/ml exceeded the targeted range.

Gentamicin Sulfate in 0.9% Saline Solution

The solubility, pH, and osmolality of gentamicin sulfate solutionsprepared with 0.9% saline solution were determined. The solubility wasdetermined by visual assessment. Only three concentrations of gentamicinwere investigated (20, 40, and 80 mg/ml).

Table 2 lists the parameters measured for gentamicin solutions and theobservations recorded during manufacture.

TABLE 2 Osmolality and pH of Gentamicin Sulfate in 0.9% Saline Weight ofActive Gentamicin Concentration Sulfate Dispensed Osmolality (mg/ml)(mg/ml) pH (mOsmol/kg) 20 34 4.94 318 40 68 4.72 353 80 136 4.82 445

Example 2

This Example involves developing the freeze-drying cycle for theclinical manufacture of the Vancomycin HCl lyophilisate. A 120 mg, 240mg, and 480 mg of Vancomycin HCl/vial strength were investigated.

Materials/Equipment

Materials

-   -   Vancomycin hydrochloride, USP, Alpharma—Denmark    -   ISO 6R clear type I glass vials, Nuova Ompi—Italy    -   20 mm freeze-drying stoppers, West Pharmaceutical Service—USA    -   20 mm flip-off caps, Capsulit S.p.A.—Italy    -   13 mm freeze-drying stoppers, West Pharmaceutical Service—USA    -   13 mm flip-off caps, West Pharmaceutical Service—USA

Equipment

-   -   Glassware for Vancomycin solution before and after filtration        (bottles).    -   Pressure vessel, Sartorius—Germany    -   Balance to check the filling weight (10 mg sensitivity),        Sartorius—Germany    -   Digital pH meter, Mettler Toledo—Switzerland    -   Karl Fischer automatic titrator DL38, Mettler        Toledo—Switzerland.    -   0.22 μm sterilizing PVDF filter, Pall    -   Manual doser, Hirschmann    -   Isolator, E.Co.Tec—Italy    -   Lyophilizer, BOC Edwards Lyoflex 04 (or Minifast 8000) with the        following characteristics: 0.4 m2 (or 0.8 m2) shelf surface;        temperature range −50° C. to 50° C.; PT 100 temperature probes;        Pirani gauge for vacuum monitoring; coil condenser with ice        capacity of 8 kg; condenser coil inlet temperature to −60° C.,        stainless steel trays with a thickness of about 2 mm;        semiautomatic crimping machine (Flexseal—Denmark)    -   DSC Pyris Diamond—USA

Composition

Solubility Study

The solubility of the Vancomycin HCl has been evaluated in order toestablish a suitable formulation to obtain a final lyophilised productwhich matches all the criteria required by its use as pharmaceuticalform.

The solubility coupled with a pH evaluation of Vancomycin HCl solutionsat different concentration was the first step to focus the suitablefinal formulation for a better development of the lyophilization cycle.

A saturated solution of Vancomycin HCl in water for injection wasprepared by adding under stirring the active agent to the solvent.

At first, the solution was clear with the solid suspended as anagglomerate; after the solid worked as crystallization nucleus and a newprecipitation occurred; so the solutions became white and more viscousbecause the solid partially swells.

Suspension was stirred for 48 h in order to reach the equilibriumconditions for the dissolution.

Suspensions was filtered first through a paper filter and then through a0.45 μm PVDF filter discarding the first drops of solution which couldhave been diluted because of the binding of the product to the membrane.

The resulting solution obtained after the two filtrations was stored at2-8° C. in order to evaluate if precipitation of the solid occurs.

The filtrated solutions of Vancomycin HCl in water coming from therespective saturated solutions, was diluted to reach a finalconcentration which gave an Abs value at λ=280 nm included into thecalibration curve.

Each diluted solution was analysed in triplicate with UV at λ=280 nm.For each solution the absorbances have been mediated and the final valuehas been substituted in the respective calibration curve equation tocalculate the concentration.

The maximum solubility of Vancomycin HCl in water is 140.9 mg/mL.

pH of Vancomycin HCl Solution in Water

Besides the solubility evaluation it was also measured the pH anddensity of Vancomycin HCl solutions at different concentrations whichcould have been taken into account for the development of theformulation and of the lyophilization cycle.

Solution Concentration Density (mg/mL) pH (g/mL) 140.8 3.4-3.5 1.046130.5 3.5-3.7 1.042 120.26 3.6-3.8 1.037 110.16 3.7-3.9 1.034 100.123.8-4.1 1.027

The pH varied within a restricted range for each concentration and theoverall pH within 140 mg/mL and 100 mg/mL was stable around the acidvalue.

Formulation

Vancomycin hydrochloride was dissolved in water for injection to form100 mg/ml formulations in 1.00 ml and 1.20 ml amounts, as shown below.

Quantity Ingredients Amount/ml Amount/Unit Vancomycin HCl 100.00 120.00mg Water for injection to 1.00 ml to 1.20 ml

Vancomycin hydrochloride was also dissolved in water for injection toform 120 mg/ml formulations in 1.00 ml, 2.00 ml, and 4.00 ml amounts, asshown below.

Quantity Amount/Unit Ingredients Amount/ml 120 mg/vial 240 mg/vial 480mg/vial Vancomycin  120.0 120.00 mg 240.00 mg 480.00 mg HCl Water for to1.00 ml to 1.00 ml to 2.00 ml to 4.00 ml injection

DSC Studies

DSC was performed on the ready to fill solution with a concentration of100 mg/ml and 120 mg/ml.

The DSC runs were performed by cooling the samples to −50° C. at acooling rate of 1° C./min, and by heating them back to 20° C. atdifferent scan rates after a period of few minutes of isothermal step.

Samples amount ranged approximately from 1 to 3 mg.

All the peaks corresponding to the detected thermal events werecalculated as onset temperature.

The DSC studies showed that there was a main event of crystallizationduring freezing and that there is no evidence of smaller crystallizationevents. These phenomena seem to indicate an absence of amorphous phaseduring freezing and a complete retention of crystalline structure byvancomycin, as confirmed by the lack of glass transitions events duringthe heating steps in all cases.

As expected, crystallization peak was displaced to lower temperatureswhen increasing the weight of the sample or the concentration of thesolution.

However no significant difference was detected among the differentconcentrations.

Detected differences are more linked to the internal variability ofsamples.

A freezing end temperature of −45° C. as well as a freezing rate of 1°C./min was chosen to ensure a full crystalline state of the VancomycinHCl during freezing.

Since the maximum allowable product temperature during initial primarydrying was −25° C., the pressure during primary drying was within ¼ to ½of the vapor pressure of ice at −25° C. Vapor of ice at −25° C. is 630μbar. The average of the thresholds, 230 μbar, was selected as themaximum allowed chamber pressure for primary drying.

Manufacturing Process

Water for injection was weighed out in a glass container on calibratedbalances.

Vancomycin HCl was added under stirring; the solution was agitated untilvancomycin was completely dissolved and the dissolution time wasrecorded.

Then, water for injection was added until the required final amount wasreached.

On the final solution, pH and density were measured and appearance wasevaluated.

The solution was filtered through a 0.22 μm PVDF membrane.

The vials were washed with distilled water and dried in an oven at 120°C. for 2 h.

The filling was performed by mass and the in process controls werecarried out by weighing the filled vials every 20 vials.

After lyophilization the following analyses were performed on the finalproduct:

water content by Karl Fischer titration; appearance of the cake,reconstitution time, appearance/clarity, pH after reconstitution.

RP-HPLC was run to confirm processing did not influence purity ofvancomycin.

Twenty (20) ml of reconstituted drug product were passed through thesterility testing membrane to confirm formulation compatibility.

Example 2A

After evaluation of the DSC results, the following lyophilization cyclenominal parameters were planned for use on the 100 mg/ml solution:

Temperature Step N^(o) Description (° C.) Pressure Time (hh:mm) 1 Load20 Atmospheric NA 2 Product freezing  20→−45 Atmospheric 01:05 3 Freezesoak time −45  Atmospheric 03:00 4 Evacuation −45   100 μbar 00:01 5Primary drying −45→ 10  100 μbar 08:00 6 Primary drying 10  100 μbar14:00 7 Secondary drying  10 → 40  100 μbar 00:30 8 Secondary drying 40 100 μbar 09:00 9 Pre-aeration 0.95 bar NA 10 Stoppering 0.95 bar NA 11Aeration Atmospheric NA Total length 35:36

The freezing soak and primary drying times were shortened with respectto the set lyophilization program.

Actually, the product reached −45° C. after 80 minutes of the freezingsoak step. It was been kept at −45° C. one hour more and then the vacuumwas pulled in the chamber to start primary drying.

During step 6 (primary drying), all the product temperature probesreached the temperature of the shelves (10° C.) after 450 minutes.

The product was left at 10° C. for 1 hour; afterwards several pressureraise tests were performed to evaluate the sublimation rate. Thepositive results of these tests allowed to start heating to 40° C. forsecondary drying. Step 6 lasted 510 minutes instead of 840 minutes.

Total length of the cycle was 29 hours.

The cake had a cohesive structure that prevented loss of friablematerial from the container during sublimation; lyophilised product wasnot really elegant because of some cracks in the cake (see the picture1).

Example 2B

In this Example involving 100 mg/ml solution, 6R vials were used. Inthis regard, twenty (20) mm neck vials enable a faster sublimation thanthe 13 mm neck vials.

An intermediate step at 0° C. during the primary drying was inserted tohave slower water vapor flow during sublimation. In this way less cracksin the lyophilization cake were observed.

The secondary drying temperature was reduced from 40° C. to 30° C.according a client's request.

Final primary drying temperature was increased from 10° C. to 15° C. totry to maintain the total length of the cycle to about 29 hours.

The nominal lyophilization parameters for this Example were:

Temperature Step N^(o) Description (° C.) Pressure Time (hh:mm) 1 Load20 Atmospheric NA 2 Product freezing  20→−45 Atmospheric 01:05 3 Freezesoak time −45  Atmospheric 03:00 4 Evacuation −45   100 μbar 00:01 5Primary drying −45→ 0   100 μbar 04:00 6 Primary drying  0  100 μbar02:00 7 Primary drying  0 → 15  100 μbar 02:00 8 Primary drying 15  100μbar 10:00 9 Secondary drying 15 → 30  100 μbar 00:15 10 Secondarydrying 30  100 μbar 09:00 11 Pre-aeration 0.95 bar NA 12 Stoppering 0.95bar NA 13 Aeration Atmospheric NA

The lower secondary drying temperature did not allow the product tomaintain a relatively low residual moisture. The overall average valuewas 3.61 wt %, while the average moisture content of previous batch was1.71 wt %.

Example 2C

In this Example involving 100 mg/ml solution, the pressure in thechamber was increased from 100 μbar to 200 μbar; a higher pressure willfavor the thermal exchanges at the gas/product interface and the thermalconductivity from the shelf to the tray. The bigger amount of heattransported to the product should result in a rise of producttemperature and consequently in a faster ice sublimation.

Furthermore, after the evaluation of the lyophilization printout, 4hours were cut from the primary drying and added the secondary dryingstep.

This Example involved the following nominal parameters:

Temperature Step N^(o) Description (° C.) Pressure Time (hh:mm) 1 Load20 Atmospheric NA 2 Product freezing  20→−45 Atmospheric 01:05 3 Freezesoak time −45  Atmospheric 03:00 4 Evacuation −45   200 μbar 00:01 5Primary drying −45→ 0   200 μbar 04:00 6 Primary drying  0  200 μbar02:00 7 Primary drying  0 → 15  200 μbar 02:00 8 Primary drying 15  200μbar 06:00 9 Secondary drying 15 → 30  200 μbar 00:15 10 Secondarydrying 30  200 μbar 13:00 11 Pre-aeration 0.95 bar NA 12 Stoppering 0.95bar NA 13 Aeration Atmospheric NA Total length 31:21

Secondary drying was shortened from the programmed 780 minutes to 450minutes. Actually, the product temperature matched the shelf temperaturevery soon due to the better heat exchange by drying at 200 μbar.

The total length of the cycle was 24.5 hours.

Average moisture content was 1.82 wt %.

The lyophilization product still showed cracks in the cake.

Example 2D

This Example involves a filling solution of 120 mg/ml to allow doses of120 mg, 240 mg, and 480 mg per vial.

All three fill volumes were lyophilized using the cycle for the largerfill sample without paying attention to a possible over drying of thelower fill volume samples.

The vancomycin 120 mg/mL filling solution was investigated by performinga scansion with the differential calorimeter, and it has been verifiedthat the main thermal events were very close to the ones detected on the100 mg/mL filling solution.

This meant that the same lyophilization cycle conditions were used forthe 100 mg/mL could be applied to the 120 mg/mL.

New holding time studies were also performed on the 120 mg/mLconcentration. The new cycle was tested on the 480 mg/vial presentationthat had the higher fill volume: 4 mL/vial.

The following nominal parameters were tested:

Temperature Step N^(o) Description (° C.) Pressure Time (hh:mm) 1 Load20 Atmospheric NA 2 Product freezing  20→−45 Atmospheric 01:05 3 Freezesoak time −45  Atmospheric 02:00 4 Evacuation −45   200 μbar 00:01 5Primary drying −45→ 0   200 μbar 04:00 6 Primary drying  0  200 μbar02:00 7 Primary drying  0→ 15  200 μbar 02:00 8 Primary drying 15  200μbar 24:00 9 Secondary drying 15 → 30  200 μbar 00:15 10 Secondarydrying 30  200 μbar 15:00 11 Pre-aeration 0.95 bar NA 12 Stoppering 0.95bar NA 13 Aeration Atmospheric NA Total length 50:21

An overall average moisture content value of 0.97 wt % was found by KarlFisher titration.

Example 2E

Following the evaluation of the product temperature profile versus theshelves temperature, the following run was cut three to four hours inthe primary drying step and four hours in the secondary drying.

The 120 mg and the 240 mg units were placed in the lyophilizer duringthe 480 mg cycle to check if over drying will affect the chemicalstability of the 120 mg and 240 mg vials.

The average residual moisture was 0.97 wt % for the 4 ml fill, 1.23 wt %for the 2 ml fill, and 1.34 wt % for the 1 ml.

The lyophilization cycle had a total length of nearly 42 hours.

Temperature Step N^(o) Description (° C.) Pressure Time (hh:mm) 1 Load20 Atmospheric NA 2 Product freezing  20→−45 Atmospheric 01:05 3 Freezesoak time −45  Atmospheric 02:00 4 Evacuation −45   200 μbar 00:01 5Primary drying −45→ 0   200 μbar 04:00 6 Primary drying  0  200 μbar02:00 7 Primary drying  0→ 15  200 μbar 02:00 8 Primary drying 15  200μbar 20:00 9 Secondary drying 15 → 30  200 μbar 00:15 10 Secondarydrying 30  200 μbar 11:00 11 Pre-aeration 0.95 bar NA 12 Stoppering 0.95bar NA 13 Aeration Atmospheric NA Total length 42:21

All three presentations had cake with a very cohesive structure even ifsome cracks were present.

Analytical Results

In Process Controls

Results Process step Analytical Test 2A 2B 2C 2D 2E Formulated pH 3.903.86 3.83 3.69 3.72 Bulk Density (g/mL) 1.027 1.028 1.030 1.0394 1.0389solution

Tests on Freeze-Dried Drug Product

Results Process step Analytical Test 2A 2B 2C 2D 2E Final Water content1.71 3.61 1.82 0.97 See Lyophilizate by KF [% w/w] below Visual aspectof Con- Con- Con- Con- Conform the cake form form form formReconstitution ~30″ ~30″ ~30″ ~5″ See below time Appearance of Con- Con-Con- Con- Conform reconstituted form form form form solution (water, 50mg/ml) pH 3.54 3.53 3.54 3.30 See below % Vancomycin 93.0 92.9 92.9 92.0See B HCl [HPLC] below % impurities 7.0 7.2 6.9 8.0 See below

Moisture Content (K.F.) [wt %]

Results 2E Sample 2A 2B 2C 2D 120 mg 240 mg 480 mg Front sample 2.063.54 1.92 1.02 1.41 1.26 0.96 Middle sample 1.40 3.12 1.80 1.01 1.271.15 0.97 Back sample 1.68 4.17 1.74 0.92 1.27 1.27 0.97 Average 1.713.61 1.82 0.97 1.34 1.23 0.97

pH

Results 2E Sample 2A 2B 2C 2D 120 mg 240 mg 480 mg Front sample 3.493.54 3.56 3.32 3.36 3.39 3.31 Middle sample 3.57 3.52 3.52 3.29 3.363.38 3.33 Back sample 3.58 3.52 3.54 3.30 3.37 3.39 3.31 Average 3.543.53 3.54 3.30 3.36 3.39 3.32

Content Vancomycin B Hydrochloride (% VMB)

Results Refer- ence Std. Sam- Vanco- ple mycin 2A 2B 2C 2E 2D Front 93.792.9 92.9 92.9 93.7 120 mg 91.3 sam- 240 mg 91.6 ple 480 mg 92.5 Mid-93.0 93.0 93.0 92.1 120 mg 91.8 dle 240 mg 91.7 sam- 480 mg 92.1 pleBack 93.1 93.0 93.0 91.9 120 mg 92.1 sam- 240 mg 91.2 ple 480 mg 91.8Aver- 93.0 92.9 92.9 92.0 120 mg 91.7 age 240 mg 91.5 480 mg 92.1 Stan-0.104 0.0327 0.0327 0.108 120 mg 0.415 dard 240 mg 0.261 Dev. 480 mg0.343 % 0.112 0.0352 0.0352 0.117 120 mg 0.453 RSD 240 mg 0.285 480 mg0.372

Related Substances (% Impurities)

Results Reference Std. Sample Vancomycin 2A 2B 2C 2D 2E Front sample 6.37.1 7.2 6.9 6.3 120 mg 8.8 240 mg 8.4 480 mg 7.5 Middle 7.1 7.3 6.9 7.9120 mg 8.2 sample 240 mg 8.3 480 mg 7.9 Back sample 6.9 7.1 7.0 8.1 120mg 7.9 240 mg 8.8 480 mg 8.3 Average 7.0 7.2 6.9 8.0 120 mg 8.3 240 mg8.5 480 mg 7.9 Standard 0.12 0.09 0.013 0.11 120 mg 0.42 Dev. 240 mg0.26 480 mg 0.42 % RSD 1.7 1.2 0.23 1.3 120 mg 5.0 240 mg 3.1 480 mg 5.3

Reconstitution Time

Reconstitution time measurement was carried out adding:

-   -   1.0 mL of WFI to the 120 mg/vial strength    -   2.0 mL of WFI to the 240 mg/vial strength    -   4.0 mL of WFI to the 480 mg/vial strength

The observed reconstitution time on the product of Example 2E was quiteshort relative to all the tested vials; about 10 seconds were needed tocompletely reconstitute the 120 mg freeze-dried drug product; 10 to 15seconds were needed to completely reconstitute the 240 mg/vialpresentation, while about 20 seconds were needed to completelyreconstitute the 480 mg units.

The reconstituted solution had a clear, light pinkish appearance and wasparticle free.

Compatibility with Sterility Testing Membrane

20 mL of reconstituted drug product were passed through the sterilitytesting membrane to confirm the formulation compatibility.

The solution passed through the filter membrane, and 17 ml of the 20 mlwere collected below the membrane.

Example 3 Summary

This Example involves a freeze-drying cycle for a 600 mg of VancomycinHCl/vial strength.

Materials/Equipment

Materials

-   -   Vancomycin hydrochloride, USP, Alpharma—Denmark    -   ISO 6R clear type I glass vials, Nuova Ompi—Italy    -   20 mm freeze-drying stoppers, West Pharmaceutical Service—USA    -   20 mm flip-off caps, Capsulit S.p.A.—Italy    -   13 mm freeze-drying stoppers, West Pharmaceutical Service—USA    -   13 mm flip-off caps, West Pharmaceutical Service—USA

Equipment

-   -   Glassware for Vancomycin solution before and after filtration        (bottles).    -   Pressure vessel, Sartorius—Germany    -   Balance to check the filling weight (10 mg sensitivity),        Sartorius—Germany    -   Digital pH meter, Mettler Toledo—Switzerland    -   Karl Fischer automatic titrator DL38, Mettler Toledo—Switzerland    -   0.22 μM sterilizing PVDF filter, Pall    -   Semiautomatic filling machine, Flexicon PF6—Denmark    -   Isolator, E.Co.Tec—Italy        -   Lyophilizer, BOC Edwards Lyoflex 04 (or Minifast 8000) with            the following characteristics: 0.4 m² (or 0.8 m²) shelf            surface, shelf temperature range was −50° C. to +50° C., PT            100 temperature probes, Pirani gauge for vacuum monitoring,            coil condenser with ice capacity of 8 kg, condenser coil            inlet, temperature arrives to −60° C., stainless steel trays            with a thickness of about 2 mm        -   Semiautomatic crimping machine, Flexseal—Denmark

Formulation

Vancomycin hydrochloride was dissolved in water for injection to form a120 mg/ml formulation, as shown below.

Quantity Ingredients Amount/ml Amount/Unit Vancomycin HCl 120.00 600.00mg Water for injection to 1.00 ml to 5.00 ml

Manufacturing Process

Water for injection was weighed out in a glass container on calibratedbalances.

Vancomycin HCl was added under stirring; the solution was agitated untilvancomycin was completely dissolved and the dissolution time wasrecorded.

Then, water for injection was added until the required final amount wasreached.

On the final solution, pH and density were measured and appearance wasevaluated.

The solution was filtered through a 0.22 μm PVDF membrane.

The vials were washed with distilled water and dried in an oven at 120°C. for 2 h.

The filling was performed by mass and the in process controls werecarried out by weighing the filled vials every 20 vials.

After lyophilization the following analyses were performed on the finalproduct:

-   -   water content by Karl Fischer titration;    -   appearance of the cake,    -   reconstitution time,    -   appearance/clarity,    -   pH after reconstitution.

RP-HPLC was run to confirm processing didn't influence purity ofVancomycin.

Lyophilization Cycle

The product was freeze-dried according the following nominallyophilization cycle parameters:

Temperature Step N^(o) Description (° C.) Pressure Time (hh:mm) 1 Load20 Atmospheric NA 2 Product freezing  20→−45 Atmospheric 01:05 3 Freezesoak time −45  Atmospheric 02:00 4 Evacuation −45   200 μbar 00:01 5Primary drying −45→ 0   200 μbar 04:00 6 Primary drying  0  200 μbar02:00 Primary drying  0→ 15  200 μbar 02:00 Primary drying 15  200 μbar24:00 7 Secondary drying 15 → 30  200 μbar 00:15 8 Secondary drying 30 200 μbar 15:00 9 Preaeration 0.95 bar NA 10 Stoppering 0.95 bar NA 11Aeration Atmospheric NA Total length 50:21 (without stoppering)

Results

An overall average moisture content value of 1.04 wt % was found by KarlFisher titration.

Cakes had a very cohesive structure even if some cracks were present.

Analytical Results

In Process Controls

Process step Analytical Test Results Formulated pH 3.69 Bulk Density(g/mL) 1.0384 solution Concentration (UV) 116.88 mg/mL

Tests on Freeze-Dried Drug Product

Process step Analytical Test Results Final Water content by KF 1.04% w/wLyophilizate Visual aspect of the cake Whitish solid compact massReconstitution time 30 seconds Appearance of Clear, colourless solutionreconstituted solution (water, 50 mg/ml) pH 3.44 % Vancomycin B by 93.3%RP-HPLC

Moisture Content (K.F.)

Sample Moisture Sample 1 (back) 1.07% Sample 2 (middle) 1.02% Sample 3(front) 1.02% Overall average 1.04%

HPLC Assay (% Vancomycin B)

Sample Vancomycin B Sample 1 (back) 93.3% Sample 2 (middle) 93.3% Sample3 (front) 93.3% Overall average 93.3%

pH

Sample pH Sample 1 (back) 3.45 Sample 2 (middle) 3.44 Sample 3 (front)3.44 Overall average 3.44

Reconstitution Time

About 30 seconds were needed to completely reconstitute the freeze-drieddrug product with 5.0 mL of WFI. The reconstituted solution had a clear,colorless appearance and was particle free.

Example 4 Summary

Nebulization characteristics of gentamicin and vancomycin solutions wereevaluated as a function of solution strength, nebulizer fill volume, andsaline concentration. Key aerosol attributes measured were emitted doseand particle size distributions. All experiments were performed usingAerotech II jet nebulizers operated continuously at 8 LPM. Forgentamicin solutions in WFI, the range of solution strengths varied from40 to 120 mg/ml, and fill volumes ranged from 2 to 4 ml. The resultingaerosol dose emitted over 30 minutes of nebulization was found to varyfrom 40 mg to over 300 mg, with the dose increasing proportionally withincreasing fill volume and solution strength. Emitted dose measurementsfor vancomycin were performed for solutions in normal saline, in 0.45%saline, and in water for injection. The range of solution concentrationstested ranged from 60 mg/ml to 140 mg/ml. The cumulative aerosol doseemitted for a 30 minute nebulization period varied from about 50 mg toover 300 mg, with the dose increasing proportionally with solutionstrength and fill mass.

Particle size distributions were measured for the above drug solutionsusing a laser diffraction spectrometer. The median particle size for allsolutions tested was in the range 2-3 μm, well within the respirablesize range. Particle size distributions for these antibiotic drugs werefound to be relatively insensitive to solution strength and fill volume.Follow-on measurements with drug and normal saline solutions indicatedthat the size distribution of nebulized antibiotics were comparable tothat for the normal saline solution.

Combined together, the above results indicate that a broad range ofaerosol doses in the respirable range may be achieved for nebulizedvancomycin and gentamicin by suitably selecting nebulizer fill volumeand solution strengths.

Objectives

To determine the amount of drug aerosol emitted during the nebulizationof gentamicin and vancomycin solutions, as a function of nebulizer fillvolume and solution strength.

To determine the size distribution of aerosols produced during thenebulization of gentamicin, vancomycin, and saline solutions as afunction of nebulizer fill volume and solution strength.

Introduction

This Example involves assessing nebulization characteristics such as theemitted dose and droplet size distribution for antibiotic drug solutionsof different strengths and at different nebulizer fill volumes. Theemitted dose information is useful in selecting solution strengths andfill masses to deliver a chosen target dose. The particle sizeinformation is useful in determining whether the aerodynamic size of theaerosol produced is in the range required for effective lung deposition(1-5 μm). Results for a placebo solution (i.e., normal saline) are alsoreported for comparison. All of the experiments were performed using anAerotech II jet nebulizer operated continuously at a nominal flow rateof 8 LPM. Aerosol emitted dose was estimated by using filters to collectthe aerosol output generated by the nebulizer, and assaying the amountof drug deposited. Particle size distributions of the generated aerosolwere measured using a Sympatec laser diffraction spectrometer.

Study Design

Characterization of Emitted Dose

For the case of gentamicin solution in water, a full factorialexperiment was performed to characterize emitted mass of aerosol as afunction of two factors, i.e. nebulizer fill volume and fill mass. Therange of solution strengths and fill volume was chosen to provide abroad range of target doses achievable with a nebulization time of 30minutes.

The test matrix for this experiment is presented in Table 1. Gentamicinsolution strength (based on mass of drug) was varied from 40 mg/ml to120 mg/ml, while the nebulizer fill volume was varied from 2 to 4 ml.Each of the 9 treatment combination was repeated twice, for a total of18 runs. The gentamicin solutions were prepared in water for injection(WFI), and were preservative free.

For the case of vancomycin, the emitted mass of aerosol wascharacterized for following three cases:

-   -   Vancomycin in normal saline, solution strength of 60 mg/ml,        nebulizer fill volume ranging from 2-4 ml.    -   Vancomycin in 0.45% saline, solution strength ranging from 60-90        mg/ml, nebulizer fill volume ranging from 2-4 ml.    -   Vancomycin in WFI, solution strength ranging from 60-140 mg/ml,        nebulizer fill volume ranging from 2-4 ml.

In the case of vancomycin, addition of salt to the formulation allowsfor tuning of solution properties such as osmolality. Test matrices forthe above three experiments are presented in Tables 2-4.

TABLE 1 Test Matrix for Gentamicin Solution in WFI Solution StrengthPattern Fill Volume [ml] [mg/mL] 13 2 120 31 4 40 22 3 80 12 2 80 11 240 21 3 40 21 3 40 13 2 120 23 3 120 31 4 40 33 4 120 11 2 40 22 3 80 122 80 23 3 120 33 4 120 32 4 80 32 4 80

TABLE 2 Test Matrix for Vancomycin Solution (60 mg/ml) in Normal SalineVancomycin at 60 mg/ml (in normal saline) Fill volume 2 ml Fill volume 3ml Fill volume 2 ml Fill volume 4 ml Fill volume 4 ml Fill volume 3 mlFill volume 3 ml Fill volume 4 ml Fill volume 2 ml

The responses measured for all of the above experiments included:

(i) the mass of drug delivered in 15 mins

(ii) the cumulative mass of drug delivered in 30 mins, and

(iii) the mass of drug remaining in the nebulizer after 30 mins ofoperation.

TABLE 3 Test Matrix for Vancomycin Solution in 0.45% Saline Fill VolumeSolution Strength Pattern [ml] [mg/L] 11 2 60 13 2 90 13 2 90 11 2 60 324 75 23 3 90 12 2 75 21 3 60 23 3 90 32 4 75 21 3 60 33 4 90 12 2 75 334 90 22 3 75 31 4 60 31 4 60 22 3 75

TABLE 4 Test Matrix for Vancomycin Solution in WFI Fill Solution VolumeStrength Pattern [ml] [mg/ml] 11 2 60 13 2 140 13 2 140 11 2 60 32 4 10023 3 140 12 2 100 21 3 60 23 3 140 32 4 100 21 3 60 33 4 140 12 2 100 334 140 22 3 100 31 4 60 31 4 60 22 3 100

Characterization of Particle Size Distribution

For the case of gentamicin solution in water, a full factorialexperiment experiment was performed to characterize the particle sizedistribution of aerosol as a function of two factors, i.e. nebulizerfill volume and fill mass. The test matrix for this experiment ispresented in Table 5. Gentamicin solution strength (based on mass ofdrug) was varied from 40 mg/ml to 120 mg/ml, while the nebulizer fillvolume was varied from 2 to 4 ml. The 9 treatment combinations were runin a random order. A fresh nebulizer was used for each run. Thenebulizers in this experiment were prequalified using a flow rate testto minimize variability in the test results.

TABLE 5 Test Matrix for Gentamicin Solution in WFI Solution Fill VolumeStrength Run Pattern [mL] [mg/mL] 1 31 4 40 2 32 4 80 3 21 3 40 4 23 3120 5 22 3 80 6 13 2 120 7 11 2 40 8 33 4 120 9 12 2 80

For the case of vancomycin, the emitted mass of aerosol wascharacterized for following three cases:

-   -   Vancomycin in normal saline, solution strength of 60 mg/ml,        nebulizer fill volume ranging from 2-4 ml.    -   Vancomycin in 0.45% saline, solution strength ranging from 60-90        mg/ml, nebulizer fill volume ranging from 2-4 ml.    -   Vancomycin in WFI, solution strength ranging from 60-140 mg/ml,        nebulizer fill volume ranging from 2-4 ml.

The test matrices for the above three experiments are presented inTables 6-8. A fresh nebulizer was used for each run. The nebulizers inthese experiments were pre-screened using a flow rate test to minimizevariability in the test results.

TABLE 6 Test Matrix for Vancomycin Solution (60 mg/ml) in Normal SalineSolution Fill Volume Strength Run Pattern [mL] [mg/mL] 1 1 2 60 2 3 4 603 2 3 60

TABLE 7 Test Matrix for Vancomycin Solution in 0.45% Saline SolutionFill Volume Strength Run Pattern [mL] [mg/mL] 1 12 2 75 2 31 4 60 3 22 375 4 33 4 90 5 13 2 90 6 11 2 60 7 32 4 75 8 21 3 60 9 23 3 90

TABLE 8 Test Matrix for Vancomycin Solution in WFI Solution Fill VolumeStrength Run Pattern [mL] [mg/mL] 1 33 4 140 2 22 3 100 3 13 2 140 4 122 100 5 11 2 60 6 32 4 100 7 21 3 60 8 23 3 140 9 31 4 60

A follow on experiment was performed to characterize particle sizedistributions of aerosols generated using vancomycin and gentamicinsolutions in water at a fixed solution strength of 120 mg/ml, and afixed fill volume of 5 ml. Particle size distributions of drug aerosolwere compared against those obtained by nebulizing normal salinesolution at a fill volume of 5 ml. The test matrix for this follow onexperiment is presented in Table 9. Each treatment was repeated 3 times.

TABLE 9 Test Matrix for Evaluation of Drug and Placebo Solutions FillVolume Run Drug [mL] 1 Normal Saline 5 2 Vancomycin 5 3 Gentamicin 5 4Gentamicin 5 5 Normal Saline 5 6 Gentamicin 5 7 Vancomycin 5 8Vancomycin 5 9 Normal Saline 5

Equipment and Materials

Equipment

-   -   Sympatec HELOS Magic BFS laser diffraction spectrometer, Ser.        No. 085    -   Mass flow meter (TSI 4000 series)    -   Rotameter    -   Volumetric flow meter, Dry Cal    -   Pressure regulator    -   Flow regulating valve    -   Flow shut-off valve    -   Pipet

Materials

-   -   Aerotech II Nebulizer    -   Tee connector and mouthpiece from Hudson RCI MicroMist Nebulizer        (Cat No. 1882)    -   Inspiratory filter (PARI electret filter)    -   Filter holder    -   One way valve    -   50 ml centrifuge tubes    -   HPLC water    -   HPLC water dispenser    -   Vancomycin HCl    -   Gentamycin Sulfate

Procedure

Characterization of Emitted Dose

The nebulizer was connected to a standard “T” piece coupled to a filterholder on one end, and a flow inlet channel provided with a one-wayvalve on the other end. The filter holder supported a PARI electretfilter used to collect the aerosol dose emitted by the nebulizer.

The nebulizer was operated using clean, dry compressed air from a sourceregulated to a pressure of about 50 psig. The flow rate of air throughthe nebulizer was controlled using a rotameter and set to a nominal flowrate 8 LPM. The drug laden air from the nebulizer passed through thecollection filter into an exhaust line provided with a backup filter anda flow regulating valve, and connected to a vacuum source. The flowregulating valve was set so that the vacuum suction flow was slightlyhigher than the nebulizer output flow. A small amount of clean make upair was allowed to enter through the one way valve to make up for theflow deficit. This arrangement enabled efficient collection of thenebulizer drug output by the filter. The emitted dose experiments wereperformed with the nebulizer operating continuously at 8 LPM for a totalnebulization time of 30 minutes. The filter/filter holder were replacedwith a fresh filter/filter holder at the 15 minute point, so that theaccumulated drug output at 15 minutes and 30 minutes could be evaluated.The filter samples were placed in centrifuge tubes and rinsed with apre-determined amount of HPLC water (ranging from 30-40 ml). Residualdrug from each filter holder was also rinsed into the correspondingcentrifuge tube using some of the filter rinsate. The residual drug fromthe nebulizer was also rinsed into a 50 ml centrifuge tube using apre-determined amount of HPLC water (ranging from 30-40 ml). The drugcontent of the filter and nebulizer samples were assessed by drugspecific HPLC assays. Note that the measurement of filter and nebulizersamples permit a full mass balance to be performed for each run.

Characterization of Particle Size Distribution

Droplet size distributions for aerosolized drug and placebo solutionswere measured using the Sympatec HELOS laser diffraction spectrometer.In preparation for a run, the nebulizer was connected to the compressedair line, the flow turned on and the pressure regulator set to a drivingpressure to generate a flow rate of 8 LPM through the nebulizer. Theflow was then turned off by closing the flow shut-off valve. Next, thenebulizer was connected to a “T” piece with one port plugged, and theother port coupled to a mouthpiece. The nebulizer was then filled withdrug solution, and mounted so that nebulizer mouthpiece was alignedparallel to the nozzle of the Rodos dry powder disperser apparatusalready installed in the spectrometer. The laser diffraction system wassetup to automatically trigger when it sensed the presence of theaerosol generated by the nebulizer. Measurements were initiated byopening the shut-off valve to pressurize the nebulizer and generate theaerosol. A total of 6 particle size distribution scans were taken foreach nebulizer run, and then averaged to provide representative sizedistribution results.

Results and Discussion

Characterization of Emitted Dose

Summarized dose delivery results for the case of gentamicin solutionsare presented in FIGS. 3-5. FIG. 3 is a bar graph showing the total drugrecovered from the nebulizer and as a function of nebulizer fill volumeand solution strength. Each recovery value is the average of tworeplicate runs (run order listed in Table 1). The drug recovery was veryconsistent across solution strengths and fill volumes, varying in therange 97.1%-101.2% of fill mass, indicating that a full mass balance wasachieved from these measurements.

FIGS. 4 a and 4 b present the cumulative emitted dose of gentamicin,respectively at the 15 min and 30 min time points, as a function of fillvolume and solution strength. Again, each value reported is the averageof two replicate runs. The delivered dose was observed to increase withan increase in both fill volume and solution strength, consistent withexpectation. A comparison of these two figures shows that the collecteddose at 15 minutes was comparable to that at 30 minutes for 2 and 3 mlfill volumes, indicating that the dose emission at these fill volumesoccurred within 15 minutes. For the 4 ml fill volume, the collected doseat 30 mins was only slightly larger than the value at 15 minutes,indicating that nebulization was largely completed within the 15 minuteperiod. From this it can be concluded that fill volumes of up to 4 ml ofgentamicin solution of strengths up to 120 mg/ml can be effectivelynebulized within a duration of 30 minutes. FIG. 4 b also indicates thata gentamicin aerosol doses spanning a factor of up to 7 can be deliveredfrom the nebulizer by suitably tuning the solution strength and fillvolume within the ranges tested.

FIG. 5 presents the gentamicin dose retained by the nebulizer at the endof 30 minutes, as a function of solution strength and fill volume. Thevalues reported are averages of two replicate runs. The retained dosewas found to increase with increasing solution strength and fill volume,with a steeper increase observed with increasing solution strength.

Similar trends in emitted dose as a function of solution strength andfill volume were obtained for the case of vancomycin. Illustrativeemitted dose measurements for vancomycin are presented in FIGS. 6-8.

For the case of 60 mg/ml solution in normal saline (see Table 2), FIG. 6plots the distribution of vancomycin drug after 30 minutes ofnebulization as a function of fill volume. The plot shows the doseretained in the nebulizer and that collected at the 15 minute (filter 1)and 30 minute (filter 2) timepoint. The reported values are averagescalculated for 3 replicate runs. As with the case of gentamicin, doseemission was found to be largely completed within 15 minutes, and theaccumulated dose (i.e. filter 1+filter 2) at the end of 30 minutes wasfound to increase with fill volume.

For the case of vancomycin solutions in 0.45% saline (see test matrix inTable 3), FIG. 7 plots the cumulative emitted dose after 30 minutes ofnebulization as a function of solution strength and fill volume. Thedelivered dose was observed to increase with increasing fill volume andsolution strength, as expected. FIG. 8 plots similar results for thecase of vancomycin solutions in WFI, obtained for the test matrixpresented in Table 4.

It is clear from FIGS. 6-8 that aerosol doses of vancomycin spanning asix fold range can be obtained from the nebulizer by suitably tuning thefill volume and solution strength within the ranges tested.

Characterization of Particle Size Distribution

Representative laser diffraction particle size measurements for the caseof gentamicin solutions (test matrix of Table 5) are summarized in FIGS.9 and 10. FIG. 9 plots the volume median diameter for aerosolizedgentamicin as a function of fill volume and solution strength (testmatrix in Table 5). Each reported value was obtained by averaging 6replicate laser diffraction measurements for each nebulization run. Themeasured median particle size for all of the gentamicin solutions variedslightly in the 2-3 μm range, and appeared to be relatively insensitiveto fill volume or solution strength. In all cases, the median particlediameter was well within the “respirable size range” considered to besuitable for pulomary drug delivery (1-5 μm). FIG. 10 plots thecumulative volume weighted particle size distributions for gentamicinaerosol for all of the solution strengths and fill volumes tested. Thesize distributions obtained for these solutions were observed to varywithin a narrow range over the fill volumes and solution strengthstested. FIG. 10 also provides a measure of the spread of the aerosolsize distribution, and it was observed that a major fraction of theaerosol was within the respirable size range.

Representative particle sizing measurements for vancomycin solutions inWFI (test matrix in Table 8) are presented in FIGS. 11 and 12, and areroughly comparable to that obtained for gentamicin solutions.

FIG. 11 indicates that the volume weighted median sizes for thesevancomycin solutions were largely within the range of 2-3 μm, also wellwithin the respirable range. The spreads of the aerosol sizedistribution, shown in FIG. 12, were similar to that obtained fornebulized gentamicin.

FIGS. 13 and 14 are plots of volume median diameter for the case ofvancomycin solutions in normal saline (test matrix in Table 6), and0.45% saline (test matrix in Table 7) respectively, obtained atdifferent solution strengths and fill volumes. The size distributionswere found to be comparable to that obtained for the vancomycinsolutions in water. In general, the size distributions of vancomycinsolutions were largely insensitive to fill volume, solution strength,and saline concentration.

Finally, results from the follow-on particle sizing study with the testmatrix listed in Table 9 are presented in FIG. 15. This figure plotsvolume median diameters for solutions of vancomycin (120 mg/ml),gentamicin (120 mg/ml) and normal saline, all obtained for nebulizerfill volumes of 5 ml. For each solution, results from three nebulizerruns are provided. It is seen from this plot that the median particlesize for all three solutions were comparable and were in the 2-3 μmrange, well within the respirable size range.

Conclusions

The emitted dose of nebulized gentamicin and vancomycin was measured asa function of solution strength, fill volume, and saline concentration.All experiments were performed using Aerotech II jet nebulizers operatedcontinuously at 8 LPM. For gentamicin solutions in WFI, the range ofsolution strengths varied from 40 to 120 mg/ml, and fill volumes rangedfrom 2 to 4 ml. The resulting aerosol dose emitted over 30 minutes ofnebulization was found to vary from 40 mg to over 300 mg, with the doseincreasing with increasing fill volume and solution strength. Emitteddose measurements for vancomycin were performed for solutions in normalsaline, in 0.45% saline, and in water for injection. The range ofsolutions tested ranged from 60 mg/ml to 140 mg/ml. The cumulativeaerosol dose emitted over a 30 minute nebulization period varied fromabout 50 mg to over 300 mg, with the dose increasing with solutionstrength and fill mass.

Particle size distributions were measured for the above drug solutionsusing a laser diffraction spectrometer. The median particle size for allsolutions tested was in the range 2-3 μm, well within the respirablesize range. Particle size distributions for these antibiotic drugs werefound to be relatively insensitive to solution strength and fill volume.Follow-on measurements with drug and normal saline solutions indicatedthat the size distribution of nebulized antibiotics were comparable tothat for the normal saline solution.

Combined together, the above results demonstrate that a broad range ofaerosol doses in the respirable range may be achieved for nebulizedvancomycin and gentamicin by suitably selecting nebulizer fill volumeand solution strengths.

Example 5

This Example involved evaluating the potential toxicity and recoveryresulting from a 14-consecutive day, nose-only inhalation administrationof vancomycin hydrochloride (vancomycin) to CD rats.

Within 2 hours prior to usage, a vancomycin nebulizer solution having aconcentration of 120 mg/ml (based on vancomycin potency of bulkmaterial) was formed by dissolving vancomycin hydrochloride (availablefrom Alpharma, Copenhagen, Denmark) in sterile water for injection USP(available from B. Braun Medical Inc., Bethlehem, Pa.). The solution wasused to generate aerosolized vancomycin for all vancomycin exposuregroups.

Nose-only exposures were conducted in a “flow-past” cylindricalinhalation chamber placed inside a steel-framed Plexiglas secondarycontainment box. The chamber contained 48 animal ports, each compatiblewith a single nose-only exposure tube, aerosol concentration samplingdevice (e.g., filter), or oxygen monitor.

The total air flow through the exposure system was balanced to achieveindividual animal port flows of ˜500 mL/min (port flow approximatedbased on total chamber flow). Measured flows included sample flow rate,nebulizer flow rate, dilution flow rate (chamber make-up air), andchamber exhaust flow. The exposure chamber had a slightly higher exhaustflow rate than inlet flow rate.

Vancomycin solution was aerosolized with two Aerotech II nebulizersoperated at 20 psi driving pressure. The target aerosol Vancomycinconcentration for all exposure levels was ˜1.0 mg/L.

Aerosolized vancomycin was administered to 3 groups of male and femaleCD rats (available from Charles River Laboratories, Kingston, N.Y.) fordurations of 30 min (Low), 90 min (Mid), and 180 min (High). A controlgroup was exposed for 180 min to aerosols generated from a normal salinesolution. Groups of rats from the Control and High level 14-dayexposures were also studied following a 14-day recovery period.Endpoints included clinical observations, body weights, clinicalpathology (hematology, clinical chemistry), urinalyses, organ weights,and histopathology.

Vancomycin aerosol concentrations were 1.23±0.16, 1.25±0.12, and1.23±0.08 mg/L for the Low, Mid, and High exposure levels, respectively.Mean particle size was determined to be in the inhalable size range forrodents (2.0-2.6 μm mass median aerodynamic diameter). Mean totalinhaled doses were estimated as 23, 71 and 139 mg/kg, and mean dosesdeposited in lung were estimated as 3, 9, and 17 mg/kg for the Low, Mid,and High exposure levels, respectively.

The vancomycin exposures were well-tolerated by all groups of rats. Allrats survived to scheduled necropsy, and there were no vancomycinrelated effects noted on clinical observations. There were also novancomycin treatment related effects on body weight.

The only organ weights to show consistent vancomycin related effectswere lungs. Lung weights were statistically significantly increased byan average approximately 8, 20, 19% of control for the Low, Mid and Highexposure levels respectively.

Exposure related histopathologic findings were limited to therespiratory tract. Observations included minimal to mild nasal mucouscell hyperplasia and hypertrophy, minimal to mild pulmonary interstitialinflammation and alveolar macrophage hyperplasia with an apparentdose-response effect, lymphoid hyperplasia of the tracheobronchial andmediastinal lymph nodes, and slight laryngeal inflammation. There wassubstantial diminution of these findings after 14 days of recovery withpulmonary interstitial inflammation, alveolar macrophage hyperplasia,and nasal mucus cell hyperplasia persisting in the high dose group, butat a lesser severity overall than seen at the end of exposure. Athreshold of response was not established although the effects in thelow dose group were generally minimal.

Clinical pathology findings were generally unremarkable. The onlyvancomycin related effect on hematology was a statistically significantincrease in neutrophils at the Mid and High exposure levels. The onlyvancomycin related effect on clinical chemistry was a mild butstatistically significant increase in aspartate aminotransferase (AST)values (˜28-46%) at the Mid and High exposure levels. Neutrophil changeswere diminished after the recovery period resolved. AST observationsresolved after the recovery period. Both findings likely resulted fromthe minimal to mild pulmonary inflammation manifested in thehistopathology findings. No vancomycin related changes were seen afterexamination of serum indicators of kidney function or urinalysis.

To conclude, the findings indicate that exposure to vancomycin at theMid level and High level exposures, predominantly, caused an irritantreaction in the respiratory tract manifested by minimal to moderatemucous cell changes in the nose and minimal to mild inflammation andmacrophage hyperplasia in the lungs. Corresponding changes in neutrophiland AST values likely resulted from the pulmonary inflammatory findings.Recovery from these effects was evident, but not entirely resolved afterthe 14-day observation period. A no observed effect level was notestablished.

Example 6

This Example involved evaluating the potential toxicity and recoveryresulting from a 14-consecutive day, face mask inhalation administrationof vancomycin hydrochloride to beagle dogs.

Within 2 hours prior to usage, a vancomycin nebulizer solution having aconcentration of 120 mg/ml was formed by dissolving vancomycinhydrochloride (available from Alpharma, Copenhagen, Denmark) in sterilewater for injection USP (available from B. Braun Medical Inc.,Bethlehem, Pa.). The solution was used to generate aerosolizedvancomycin for all vancomycin exposure groups.

The exposure system consisted of a single, cylindrical, plexiglassinhalation chamber (volume of ˜23.7 L, 14.61-cm radius, 35.56-cmheight). The chamber was supplied with two Aerotech II nebulizersoperated at ˜40 psi. Nebulized test article and nebulizer air supply wasdiluted with ˜10 L/min HEPA-filtered dilution air. The flow through thesystem was ˜36 L/min.

The aerosolized vancomycin was administered via a face mask to 3 groupsof male and female beagle dogs for durations of 15 min (Low), 30 min(Mid), and 60 min (High). A control group was exposed for 60 min toaerosols generated from normal saline solution, i.e., 0.9% sodiumchloride injection USP (available from B. Braun Medical Inc.).

Groups of dogs from the Control and High level 14-day exposures werealso studied following a 14-day recovery period. Endpoints for allgroups of dogs included physical examinations, clinical observations,body weights, ophthalmology, cardiovascular EKG, clinical pathology(hematology, clinical chemistry), urinalyses, organ weights,histopathology, and toxicokinetics.

Vancomycin aerosol concentrations were 1.39±0.20, 1.51±0.19, and1.49±0.15 mg/L for the Low, Mid, and High exposure levels, respectively.Mean particle size was determined to be in the inhalable size range fordogs (1.9-2.6 μm mass median aerodynamic diameter). Mean total inhaleddoses were estimated as 10, 23, and 45 mg/kg, and mean doses depositedin lung were estimated as 2, 5, and 9 mg/kg for the Low, Mid, and Highexposure levels, respectively.

The vancomycin exposures were well-tolerated by all groups of dogs. Alldogs survived to scheduled necropsy. There were no vancomycin relatedeffects noted on physical examinations, clinical observations,ophthalmology, cardiac ECG tracings, hematology, clinical chemistry,urinalyses, gross necropsy observations, and organ weights.

Histopathology examinations of tissues revealed no effect of Vancomycinexposure in the organs and tissues examined outside of the respiratorytract. Likewise, there was an absence of microscopic alterations in thenasal cavity/turbinates, larynx, and trachea. The effects of Vancomycinexposure were limited to microscopic findings in the lung.Treatment-related increased incidence of minimal to mild chronicinterstitial inflammation, alveolar histiocytosis, and bronchial lymphnode lymphoreticular hyperplasia were observed. Among Control and Highlevel animals in the Recovery groups there were no treatment-relateddifferences in the macroscopic and microscopic findings.

In conclusion, effects of Vancomycin exposure were limited to minimal tomild pulmonary histopathology at the termination of exposure. Recoveryof histopathological effects was complete after 14 days. The minimal tomild chronic interstitial inflammation was generally comparable withbackground inflammatory changes in beagle dogs. The alveolarhistiocytosis was reflective of enhanced clearance that occurs withoutalveolar injury. The lymphoreticular hyperplasia was considered anadaptive response that facilitates lung clearance mechanisms. Sincecorresponding fibrosis and alveolar epithelial injury were notcharacteristic of the observed effects, the lung changes and relatedlymph node changes were not considered adverse effects. Based on thesefindings, the no observed adverse effect level (NOAEL) was the highexposure level corresponding to an inhaled dose of 45 mg/kg and adeposited lung dose of 9 mg/kg.

Example 7

Amikacin Sulfate sterile solution for inhalation, 125 mg/ml wasmanufactured and characterized as follows. Approximately 13.5 L ofsterile water for injection was added to a glass carboy fitted with alightning labmaster mixer. Amikacin sulfate was added to the carboy andthe solution was stirred. The solution was mixed until the entire APIwas dissolved. A sample of the solution was taken and pH measured. Withcontinued stirring, pH was adjusted with 1.0N HCl to be within 5.5-6.3with a target pH of 5.9. After pH adjustment, sufficient quantity ofsterile water for injection was added until the final weight of solutionof 21,328 g. was reached. The pH of the final solution was verified tobe within an acceptable range. The solution was then sparged withfiltered nitrogen at a rate of 1.5 L/min for 15 minutes. The solutionwas then filtered through the 0.22 micron sterile filter.

Prior to filling the solution, each vial was purged with nitrogen. Thesolution was filled by weight using a Cazzoli filler/stopper machineinto 5 ml amber vials to a target weight of 4.27±0.08 g. The vials werestoppered with 20 mm Teflon-coated stoppers and secured with aluminumflip off seals. Filled vials were stored at 2-8° C. The composition issummarized in Table A below.

TABLE A Ingredient g per batch Amikacin Sulfate 3525.0 g HydrochloricAcid qs to pH 5.9 NaOH qs to pH 5.9 Sterile Water for Injection qs to21, 328 g Nitrogen, NF Qs

Stability over time was assessed for as formulation made substantiallyas show in table A, with regard to total amikacin active, relatedsubstances, such as degradation products, appearance, pH, particulatesand sterility. Thus samples were stored at 5° C. (Table B), at 25°C./60% relative humidity (RH) (Table C), and at 40° C./75% RH (Table D).In each case samples were stored in 5 mL amber glass vials, with 20 mmTeflon stoppers and 20 mm aluminum overseals. Results of each of thesestorage conditions are shown in Tables B, C and D, respectively.

TABLE B Attributes Specification Initial 1 mo. 3 mos. 6 mos. 9 mos. 12mos. 18 mos. 90.0%-110.0% l.s. 99.8 104.6 104.0 104.3 104.9 100.0 105.599.8 100.6 Appearance Meets Test¹ MT MT MT MT MT MT MT Kanamycin @ rrtReport results 0.73 0.78 0.68 0.34 0.33 0.28 0.22 0.72 0.64 Rel.Substance A Report results 0.66 0.70 0.19 0.55 0.46 0.50 0.36 @ rrt 0.860.60 Unidentified @ rrt Report results 6.90 6.81 5.38 3.38 3.02 2.902.29 0.61 6.27 Unidentified @ rrt Report results 0.53 0.45 0.45 0.230.26 0.21 0.23 0.67 0.50 Total Related 10.0% 8.82 8.74 6.70 4.50 4.073.89 3.10 Substances 8.01 pH 5.5-6.3 5.6 5.6 5.5 5.6 5.5 5.7 5.6Particulate Matter Particles = 10 μm 50 3 7 24 38 5 2 NMT 6000 Particles= 25 μm 0 0 0 0 1 0 0 NMT 600 Sterility Meets USP Conforms NP NP NP NPMT NP

TABLE C Attributes Specifications Initial 1 mo. 3 mos. 6 mos. Assay90.0%-110.0% 99.8 104.0 105.3 101.1 I.s. 99.8 Appearance Meets Test¹ MTMT MT MT Kanamycin @ Report results 0.73 0.81 0.71 0.44 rrt 0.72 0.64Rel. Substance Report results 0.66 0.72 0.18 0.59 A @ rrt 0.86 0.60Unidentified @ Report results 6.90 6.87 5.28 3.55 rrt 0.61 6.27Unidentified @ Report results 0.53 0.50 0.49 0.28 rrt 0.67 0.50 TotalRelated 10.0% 8.82 8.90 6.66 4.86 Substances 8.01 pH 5.5-6.3 5.6 5.6 5.65.6 Particulate Particles = 50 12 18 33 Matter 10 μm NMT 6000 Particles= 0 1 0 1 25 μm NMT 600 Sterility Meets USP Conforms NP NP NP

TABLE D Attributes Specifications Initial 1 mo. 3 mos. 6 mos. Assay90.0%-110.0% 99.8 104.1 104.7 106.3 I.s. 99.8 Appearance Meets Test¹ MTMT MT clear, faint yellow solution Kanamycin @ Report results 0.73 0.991.06 1.00 rrt 0.72 0.64 Rel. Substance Report results 0.66 0.71 0.160.60 A @ rrt 0.86 0.60 Unidentified @ Report results 6.90 6.81 4.64 3.52rrt 0.61 6.27 Unidentified @ Report results 0.53 0.54 0.60 0.61 rrt 0.670.50 Total Related 10.0% 8.82 9.05 6.46 5.73 Substances 8.01 pH 5.5-6.35.6 5.6 5.5 5.5 Particulate Particles = 50 32 20 27 Matter 10 μm NMT6000 Particles = 0 1 0 1 25 μm NMT 600 Sterility Meets USP Conforms NPNP NP

FIG. 16 is a graphical representation of certain of the stability dataprovided in Tables B, C and D. In the Fig., the line marked by diamondsrepresents the 5° C. storage condition, the line marked by the squaresrepresents 25° C./60% RH storage conditions and the line marked by thetriangle represents 40° C./75% RH storage conditions. The Fig shows thatthe percentage related substances, i.e. impurities, diminishes overstorage time. It is thought that this is a function of detactability ofthe impurities. It is evident, however, that the compositions remainstable, with respect to impurities, over time.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the methods of the presentinvention can be carried out with a wide and equivalent range ofconditions, formulations, and other parameters without departing fromthe scope of the invention or any embodiments thereof.

All patents and publications cited herein are hereby fully incorporatedby reference in their entirety. The citation of any publication is forits disclosure prior to the filing date and should not be construed asan admission that such publication is prior art or that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

1-21. (canceled)
 22. A kit, comprising: a first container containing afirst aqueous solution comprising a first antibiotic; and a secondcontainer containing a second aqueous solution comprising a secondantibiotic, wherein the first antibiotic is amikacin and the secondantibiotic is the same or different, and wherein a concentration, or anamount, or both of the first antibiotic is the same as, or differentfrom a concentration, or an amount, or both of the second antibiotic.23-42. (canceled)
 43. A kit comprising: a unit dose comprising about 3to 10 ml of a preservative-free liquid composition for aerosolizationconsisting essentially of about 400 mg to about 750 mg amikacin, or saltthereof, and water; and a vibrating mesh nebulizer.
 44. The kit of claim48, wherein the concentration of amikacin is from about 110 mg/ml toabout 150 mg/ml, or a potency from about 500 μg/mg to about 1100 μg/mg,or both.
 45. The kit of claim 49, wherein the liquid composition has apH from about 4 to about
 6. 46. The kit of claim 50, wherein the liquidcomposition has an osmolarity ranging from about 90 mOsmol/kg to about500 mOsmol/kg.
 47. The kit of claim 51, wherein no precipitate forms inthe liquid composition when the liquid composition is stored for 1 yearat 25° C.
 48. A unit dose comprising: about 3 to 10 ml of apreservative-free liquid composition for aerosolization consistingessentially of about 400 mg to about 750 mg amikacin, or salt thereof,and water.
 49. The unit dose of claim 43, wherein the concentration ofamikacin is from about 110 mg/ml to about 150 mg/ml, or a potency fromabout 500 μg/mg to about 1100 μg/mg, or both.
 50. The unit dose of claim43, wherein the liquid composition has a pH from about 4 to about
 6. 51.The unit dose of claim 43, wherein the liquid composition has anosmolarity ranging from about 90 mOsmol/kg to about 500 mOsmol/kg. 52.The unit dose of claim 43, wherein no precipitate forms in the liquidcomposition when the liquid composition is stored for 1 year at 25° C.